diff --git "a/deduped/dedup_0377.jsonl" "b/deduped/dedup_0377.jsonl"
new file mode 100644--- /dev/null
+++ "b/deduped/dedup_0377.jsonl"
@@ -0,0 +1,68 @@
+{"text": "Brucella suis 1330 demonstrates that the properties of the target molecule have the potential to strongly influence the rate and extent of hybridization between transcript and tethered oligonucleotide probe in a microarray experiment.Secondary structure in the target is a property not usually considered in software applications for design of optimal custom oligonucleotide probes. It is frequently assumed that eliminating self-complementarity, or screening for secondary structure in the probe, is sufficient to avoid interference with hybridization by stable secondary structures in the probe binding site. Prediction and thermodynamic analysis of secondary structure formation in a genome-wide set of transcripts from Brucella using a freely available oligo design software package. 21 \u00b1 13% of the nucleotides in each probe binding site are within a double-stranded structure in over half of the folds predicted for the cDNA target at 65\u00b0C. The intramolecular structures formed are more stable and extensive when an RNA target is modeled rather than cDNA. When random shearing of the target is modeled for fragments of 200, 100 and 50 nt, an overall destabilization of secondary structure is predicted, but shearing does not eliminate secondary structure.Despite the relatively high hybridization temperatures and 1M monovalent salt imposed in the modeling process to approximate hybridization conditions used in the laboratory, we find that parts of the target molecules are likely to be inaccessible to intermolecular hybridization due to the formation of stable intramolecular secondary structure. For example, at 65\u00b0C, 28 \u00b1 7% of the average cDNA target sequence is predicted to be inaccessible to hybridization. We also analyzed the specific binding sites of a set of 70mer probes previously designed for Secondary structure in the target is pervasive, and a significant fraction of the target is found in double stranded conformations even at high temperature. Stable structure in the target has the potential to interfere with hybridization and should be a factor in interpretation of microarray results, as well as an explicit criterion in array design. Inclusion of this property in an oligonucleotide design procedure would change the definition of an optimal oligonucleotide significantly. Target molecules  hybridize transiently to the probe oligomers until they form stable double helices with their specific probes. At some point, the rate of on and off reactions reach equilibrium, and the concentration of the target in the sample solution can be calculated. Transcript abundance is assessed by the relative intensity of signal from each spot on the array. This interpretation of array data relies on the assumption that each hybridization reaction goes to completion within the timeframe of the experiment and that the behavior of all pairs of intended reaction partners in the experiment is somewhat uniform.Sequence-specific hybridization of a long single-stranded labeled DNA or RNA target molecule to shorter oligonucleotide probes is the basis of the gene expression microarray experiment. In this type of microarray experiment, gene specific There are three major types of DNA microarrays, which differ in the approach used for probe design: Affymetrix type microarrays , which aA number of oligonucleotide design software packages have been published in recent years, each having design strengths in one of a number of criteria -14. SeveFew of the available array design packages explicitly consider the possible structures of the transcript-derived molecules in the sample solution and their impact on whether the microarray will provide an effective assay, although the OligoDesign web server  does comBrucella suis. We have assessed the stability of structures formed in the transcript and the accessibility of the binding sites of optimal probes generated using commonly applied design criteria. Because random shearing of the full-length target molecule is used in some protocols, we have also modeled the effects of shearing to an average length on the prevalence of secondary structure in selected targets.In order to estimate the prevalence of stable secondary structure in long target molecules, and thus the impact such structures might have on the analysis of microarray data, we have modeled secondary structure formation in mRNA transcripts of the intracellular pathogen Brucella suis  is near 70% of the average for Brucella, while the average \u0394G for transcripts from the GC-poor genome (L. lactis) are even lower (30% at 52\u00b0C). However, even in the most GC-poor genome, stable target secondary structure in the single-stranded target is widespread.Our modeling results obtained for the genome-wide set of intact single-stranded DNA or RNA targets demonstrate that stable secondary structures are widespread in target mixtures from s Figure  and in rOur results demonstrate that a significant fraction of nucleotide sites in the average target mixture, whether single stranded DNA or RNA, will be found in stable secondary structure under the hybridization conditions used in oligonucleotide microarray experiments, and will be relatively inaccessible for intermolecular interactions. Figure Figure Figure in silico shearing, revealed the likely presence of stable secondary structures in both full-length target and sheared target mixtures. These structures do not convert completely to random coil with either increasing hybridization temperature, more extensive shearing, or both. These secondary structures may therefore compete with the intended target for effective probe annealing in a microarray experiment, resulting in a misinterpretation of the amount of target present in the sample.Lack of bioinformatics tools that incorporate experimentally validated biophysical properties of nucleic acids as a criterion for synthetic oligomer probe design is a major challenge for do-it-yourself microarray designers. One biophysical characteristic, which we predict will reduce the binding efficiency of microarray probes to their targets, is the propensity of long single-stranded DNA or RNA molecules to form stable secondary structure. 3-D structures such as hairpins and stacked regions have the potential to pre-empt target nucleotides, thus blocking regions of the target molecules from hybridizing to their intended probes. Prediction and thermodynamic analysis of secondary structure at a range of temperatures in full length target sequences, as well as in subsequences formed by in silico experiment, secondary structure prediction in the target is being used to develop a new criterion for oligonucleotide probe design. Our results from this modeling experiment demonstrate that the implicit assumption used until now \u2013 that eliminating probe secondary structure by avoiding self-complementarity eliminates target secondary structure as well \u2013 is valid only when the target and probe are of the same length. Use of target secondary structure as an explicit criterion will allow for masking or preferentially avoiding the regions of the target sequence in which base pairs are directly involved in secondary structure formation, to eliminate these regions from the sequence for the purpose of the search for the optimal probe.Based on the results of this In this study we have assigned accessibility scores to sites in the target sequence based only on the fraction of predicted structures within 5% of the energy optimum, in which a residue is found in a single-stranded conformation. While this measure is not too computationally intensive to compute, and can be applied to genome-scale problems using readily available software (Mfold), it is not the most physically rigorous definition of accessibility. By equally weighting each possible structure in the ensemble of optimal and suboptimal structures that a molecule can form, it is possible that secondary structure at some positions in the molecule is overcounted; bonds which form only in rare conformations are considered equal to bonds which are present in the lowest-energy structure. The program Sfold -18 assigWhen we compared MFold-based accessibility predictions for an individual transcript to those generated by SFold and RNAFold, we found that the difference in average predicted accessibility over an entire transcript is small. We computed accessibility for the transcript of human 1CAM-1, which has been mapped experimentally to determine its accessibility . The aveIn this study, we focused specifically on the DNA/RNA base pairs that are actively involved in hydrogen bond formation. We realize that other accessibility considerations will have to be added to the scoring scheme in practice. The structure of a long single stranded DNA or RNA molecule can contain many nucleotides that, while not part of a double-helical stem, remain inaccessible to hybridization due to their location inside small loops within the target secondary structure. A loop is a somewhat constrained structure as well, and the length at which it presents accessible sequence that favors hybridization has been shown to be on the order of 10 nucleotides and longer , while nB. suis example, use of a low annealing temperature, e.g. 42\u00b0C which is the temperature used in some published 70-mer array experiments [Development of a target secondary structure criterion for oligonucleotide array design is expected to impose restrictions on the probe selection beyond the sequence similarity and melting temperature criteria that are currently used, especially in cases where short probe length restricts the annealing temperature used in the hybridization protocol to 22\u201337\u00b0. In the eriments , would rWe have shown here that while shearing reduces overall \u0394G of secondary structure formation for individual molecules in the target solution, shearing does not in itself eliminate formation of secondary structure in single-stranded DNA or RNA. The question of whether shearing should be used for long oligomer arrays is still an open one. While some signal may be gained by reducing the stability of secondary structure in the target molecule, random shearing by its nature creates a mixture of targets that may have substantially different affinities. For instance, in a 300 nt transcript that is targeted by a 70mer oligonucleotide, there is nearly a one in four chance that a random break in the sequence will occur within the target site for which the probe is designed. Short fragments may present a substantially different binding site, and therefore have a different binding affinity, than the full-length transcript that is considered when the probe is designed. This is illustrated in Figure 8d, where binding of a 50mer sheared fragment to a 70mer probe leaves a dangling end in the probe. A break very close to one end or the other of the target site may create a target that still binds to the probe, though with reduced affinity; a break closer to the middle of the target site may produce fragments that bind partially to the probe, competing for binding with perfect matches.In other experimental contexts where hybridization is critical to success, the impact of secondary structure in single stranded polynucleotides on results has been recognized and is now being systematically studied (18\u201321). Intramolecular folding of mRNAs is so extensive that only 5\u201310% of most transcripts is accessible to binding of complementary nucleic acids; however the modeling of long molecules has not proven to give very accurate binding predictions -25. In fBrucella had binding sites in the target which were predicted to contain a stretch of unpaired bases of at least 14 nt in length; at 65\u00b0C, that fraction increased to 93%. Based on these findings we would expect that at 52\u00b0C only 57% of our probes would encounter optimal conditions for hybridization and therefore would demonstrate the expected behavior in the experiment, where intensity is expected to scale with target concentration. We predict that the remaining probes, which have shorter, or no, accessible sequences, will exhibit modified binding behavior, and we plan to conduct experiments to characterize this behavior. We have shown conclusively that avoiding self-complementarity in the probe when designing an oligonucleotide array is insufficient to eliminate secondary structure from the binding site in the target. By combining the procedure for systematic computational assessment of transcript accessibility described in this study with selective experimental validation of the impact of predicted accessibility on hybridization, we will develop a useful criterion for avoiding troublesome secondary structure when designing microarray targets.The results of the current study suggest a significant role for target secondary structure in hybridization to oligonucleotide arrays, which will warrant further investigation. Oligonucleotide probe binding sites in a significant fraction of transcripts are found in double-stranded conformations even in cases where self-complementarity was avoided during the probe design process. We find that at 52\u00b0C, for example, approximately 57% of probes designed for Brucella suis 1330 genome. Brucella suis has a relatively high (57%) genomic GC content. Brucella suis was chosen for this experiment because our collaborators have previously acquired a custom synthetic oligomer microarray for this organism, developed using standard oligo array design software, and we have access to both target sequences and to a set of unique probe sequences that define the interaction sites for which expression results have been obtained by the laboratory.Prediction and thermodynamic analysis of secondary structure was performed for all protein-coding gene transcripts predicted from 3264 CDSs in the Escherichia coli), and 50 from the GC-poor genome of the nonpathogenic AT-rich gram-positive bacterium Lactococcus lactis (35% genomic GC content). The Brucella suis genes ranged in length from 90 to 4,803 bp, with an average transcript length of 851 bp. The E. coli genes ranged in length from 140 to 2,660 bp, with an average transcript length of 792 bp. The range of GC content in the genes chosen was 37% to 57% with an average value of 50%, which is reasonably representative of the E. coli genome. The L. lactis genes chosen ranged in length from 140 to 2,730 bp, with an average transcript length of 765 bp., and ranged in GC content range from 30% to 42% with an average value of 35%.In order to determine whether Brucella sequences form atypical structures we randomly picked and analyzed 50 gene coding sequences from a compositionally balanced genome  using ArrayOligoSelector (pick70) [70-mer probes for each (pick70) . ArrayOlProbe and transcript secondary structure were predicted using the Mfold 3.1 software package ,29. MfolAccessibility in folded single-stranded DNA or RNA has recently begun to be addressed in a few experimental studies, mainly with the goal of targeting appropriate sites for RNAi. Because the structure of single-stranded nucleotide molecules is much more dynamic than that of proteins, with each molecule likely to exist in an ensemble of structures, and because the 3D structure of these molecules is rarely known, there is not yet a consensus representational standard of per-residue accessibility for single-stranded nucleic acids. Ding et al. ,18 impleIn this study, we chose to use the less physically rigorous approximation of probability of single strandedness as a simple fraction of predicted optimal and suboptimal structures in which a residue is found to be part of a single stranded structure, as computed by Mfold. Accessibility scores derived from MFold predictions have been used in limited studies of RNA structure focused on hammerhead ribozymes, antisenB. suis. Secondary structure and thermodynamics were computed for each of these fragments individually.Random shearing of the target mixture is an approach that is often offered as a solution for the problem of target secondary structure. The actual content of a sheared mixture of DNA or RNA fragments is complex. Shearing breaks the molecule not in predictable locations, but in random locations that give rise to a distribution of fragments around an average fragment length. In order to simulate the effects of different degrees of shearing on structure formation and stability in a transcript, we picked fragments of 200, 100, or 50 bases in length, choosing the start position via a sliding window of 10 bases. Secondary structure prediction for all fragments derived from every transcript in the B. suis genome is computationally intensive and produces an extremely large amount of output. Since our initial goal was to determine how much the method would affect the number and type of secondary structures probes would be expected to bind the shearing simulation was performed for fragments derived from the 300 bp Ure-1A gene of VGR participated in the design of the study, carried out the simulations and analysis, and drafted the manuscript. JWW participated in the design of the study and helped to draft the manuscript. CJG conceived of the study, participated in its design, coordinated the research and analysis, and drafted the manuscript."}
+{"text": "It's a basic biological principle that living things share certain fundamental traits. That's why understanding the mechanisms of cell division in single-celled yeast, say, can offer insight into cell division in humans. Now Joseph Heitman and colleagues report that the evolutionary events that spawned sex chromosomes in yeast resemble those that shaped sex chromosomes in animals.Cryptococcus, Heitman and colleagues found that this fungal sex-determining region arose via a series of discrete events that echo those that shaped mammalian sex chromosomes.Strictly speaking, yeast\u2014the common name for single-celled fungi\u2014don't have sex chromosomes; they have sex-determining regions within chromosomes, called mating type, or MAT, loci. In a comparative genomic analysis of the MAT locus in three species of the human pathogenic fungus a, for example, and mating type alpha. Still, yeast manage a measure of complexity and considerable elegance in the systems they deploy to sexually reproduce.A primary benefit of sexual reproduction is the genetic diversity gained from reshuffling genetic material during meiosis, which creates gametes. Yeast sex, such as it is, accomplishes the same thing. Of course, sexual identity for a fungus does not take the form of sperm or egg but of mating type a or alpha mating status are alleles (variants) of a single MAT locus. Cells with the MATa allele are mating type a, while cells with the MATalpha allele exhibit mating type alpha. A cell can switch its mating type when genetic exchange, or recombination, between two mating loci occurs.In ascomycetes, like baker's yeast, the MAT locus is small and includes just a few genes. The genes that determine a cell's Ustilago maydis\u2014a maize pathogen that some consider a culinary delicacy\u2014mating is more complex, and sexual identity is determined by two unlinked genomic regions with distinct classes of genes. Cells must be of different mating types at both loci to allow sexual reproduction. To their surprise, Heitman and colleagues discovered that the mating locus of Cryptococcus neoformans\u2014a basidiomycete fungus that infects humans and is associated with transplant recipients, patients with AIDS, and other immune-compromised patients\u2014exhibits several unique features, common to neither ascomycetes or their basidiomycete relatives.In basidiomycetes, like the corn smut C. neoformans locus occupies a single region and is unusually large, spanning more than 100 kilobases and containing over 20 genes, including those typically segregated in separate locations in other basidiomycetes. Like on the human Y chromosome, the sex-determining genes of C. neoformans are interspersed with non-sex-related genes. And unlike ascomycetes, which also have a single active MAT locus and two mating types, no mating type switching occurs as there are no silent mating type cassettes in the genome.Unlike most basidiomycetes, the a and alpha alleles of C. neoformans' closest relative, C. gattii, and compared these variants to four already characterized variants derived from two C. neoformans subspecies. All six MAT alleles share characteristic features, including a fairly large size, a common gene set, and dramatic genomic migration during evolution . Each MAT allele has genes with different evolutionary histories, ranging from ancient to recent, that fall into distinct patterns based on shared nucleotide composition and mating type. The patterns correlate with how long the genes have occupied the MAT locus, suggesting how it evolved.Heitman and colleagues sequenced the Cryptococcus MAT locus resembles the evolution and structure proposed for the ancient Y chromosome, the authors argue that Cryptococcus can serve as a valuable model to study the molecular dynamics of sex chromosomes.The authors hypothesize that this novel structure was formed by chromosomal rearrangements that linked two unrelated genomic regions into a single region. Recombination between these sex-determining regions was suppressed after other events blurred their boundaries. Specific genes in the once separated loci then attracted mobile elements in the genome to their sites, thus precipitating expansion of the locus. Because the"}
+{"text": "Saccharomyces cerevisiae lacks functional equivalents of most of these factors; thus, it has been unclear whether the spliceosome could effectively distinguish among transcripts. To address this question, we have used a microarray-based approach to examine the effects of mutations in 18 highly conserved core components of the spliceosomal machinery. The kinetic profiles reveal clear differences in the splicing defects of particular pre-mRNA substrates. Most notably, the behaviors of ribosomal protein gene transcripts are generally distinct from other intron-containing transcripts in response to several spliceosomal mutations. However, dramatically different behaviors can be seen for some pairs of transcripts encoding ribosomal protein gene paralogs, suggesting that the spliceosome can readily distinguish between otherwise highly similar pre-mRNAs. The ability of the spliceosome to distinguish among its different substrates may therefore offer an important opportunity for yeast to regulate gene expression in a transcript-dependent fashion. Given the high level of conservation of core spliceosomal components across eukaryotes, we expect that these results will significantly impact our understanding of how regulated splicing is controlled in higher eukaryotes as well.Appropriate expression of most eukaryotic genes requires the removal of introns from their pre\u2013messenger RNAs (pre-mRNAs), a process catalyzed by the spliceosome. In higher eukaryotes a large family of auxiliary factors known as SR proteins can improve the splicing efficiency of transcripts containing suboptimal splice sites by interacting with distinct sequences present in those pre-mRNAs. The yeast  S. cerevisiae. By systematically examining the effects of individual mutants in the spliceosome on the splicing of all substrates, we have uncovered a surprisingly complex relationship between the spliceosome and its full complement of substrates. Contrary to the idea that the spliceosome engages in \u201cgeneric\u201d interactions with all intron-containing substrates in the cell, our results show that the identity of the transcript can differentially affect splicing efficiency when the machine is subtly perturbed. We propose that the wild-type spliceosome can also distinguish among its many substrates as external conditions warrant to function as a specific regulator of gene expression.The spliceosome is a large RNA-protein machine responsible for removing the noncoding (intron) sequences that interrupt eukaryotic genes. Nearly everything known about the behavior of this machine has been based on the analysis of only a handful of genes, despite the fact that individual introns vary greatly in both size and sequence. Here we have utilized a microarray-based platform that allows us to simultaneously examine the behavior of all intron-containing genes in the budding yeast  Many eukaryotic gene transcripts are spliced; here the authors show that components of the splicing complex can distinguish between different introns in highly homologous transcripts. The coding regions of most eukaryotic genes are interrupted by introns, which must be removed for proper gene expression. The removal of introns requires single-nucleotide precision in order to faithfully convert genomic information into functional protein. The process of intron removal is performed by the spliceosome, a large ribonucleoprotein that catalyzes two sequential transesterification reactions ,2. The sTo date, the vast majority of what is known about the mechanism of pre-mRNA splicing has been deduced from experiments using, at most, a handful of transcripts. Yet it remains unknown how well these transcripts represent the behavior of the entire complement of spliceosomal substrates. In higher eukaryotes, where genes are often interrupted by multiple introns, it is known that the spliceosome can utilize specific sequences present in individual transcripts to regulate both quantitative and qualitative aspects of gene expression ,7. Two lSaccharomyces cerevisiae appears much simpler in a number of ways. Whereas more than 95% of human genes are interrupted by an intron [By comparison to higher eukaryotes, splicing in the yeast n intron , only abn intron . As seenn intron . NotablyGiven the relative simplicity and similarity of intron-containing transcripts in yeast and the limited role of the SR proteins, it has been a reasonable expectation that the yeast spliceosome would have a restricted capacity to distinguish among its different intron-containing substrates. In this case, the behavior of a few individual transcripts could be extrapolated to accurately describe the behavior of most spliceosomal substrates. The advent of splicing-specific microarrays, which facilitate a simultaneous examination of all intron-containing transcripts , allows To examine genome-wide changes in splicing in yeast, we have designed microarrays similar to those previously described  that allBecause there are multiple, independent probes describing the behavior of each intron-containing gene, analysis of a splicing-specific microarray is inherently more complicated than that of a standard expression array. In order to assess the splicing behavior of any given transcript it is important to simultaneously examine the changes in signal on all three feature types. In analyzing data from splicing-specific microarrays, others have generated splicing indices for each gene by compressing the data derived from the different feature types. For example, splicing changes can be represented as a precursor/mature (PM) index by dividing the ratio of the movement of the pre-mRNA feature by the ratio of the mature mRNA feature ,12. Althprp2-1 mutation after both were shifted to the nonpermissive temperature of 37 \u00b0C. The spliceosomal Prp2 protein is a member of the DEAD/H box family of RNA-dependent ATPases and plays an essential role in catalyzing an as yet unidentified structural rearrangement required for activation of the spliceosome prior to the first chemical step [cal step . As suchThe different behaviors displayed by these two groups of transcripts presumably reveal important differences in the complex interplay of cellular machineries that define the steady-state levels of both the precursor and mature forms of each of these transcripts. For example, the rate of precursor accumulation observed for a given transcript depends upon, among other things, the transcription rate of that gene, the degradation rate of the transcript, the rate at which the transcript is normally spliced, and the extent to which its splicing is inhibited. Likewise, the decrease in mature mRNA is a function of both the extent of splicing inhibition and the decay rate of the mature form of that transcript. For those genes marked by the red bar in prp2-1 mutation shows that precursors rapidly accumulate for about 80% of the intron-containing genes. While the detailed behaviors of the different transcripts are varied, this result appears to satisfy the simple prediction that defects in this factor would result in defective splicing of all actively transcribed genes. At least two different scenarios may explain the failure to detect precursor accumulation for the remaining 20% of the transcripts. It is almost certainly true that some of the intron-containing genes are transcriptionally quiescent during logarithmic growth conditions. Naturally, no precursor accumulation would be expected for such genes. Alternatively, these transcripts may be insensitive to the particular defect introduced by the prp2-1 mutation.By simultaneously examining each of the feature types as described above, the global splicing profile resulting from the PRP5 and PRP8. Like PRP2, PRP5 encodes an essential member of the DEAD/H box family of RNA-dependent ATPases. By comparison with Prp2, the activity of Prp5 is required to promote a molecular rearrangement earlier in the splicing pathway, catalyzing the stable addition of the U2 small nuclear ribonucleoprotein to the branch point sequence [In order to further distinguish between transcript behaviors, we examined the RNA from strains containing mutations in two additional core spliceosomal components: sequence . As withsequence ,16. As wsequence ,18.prp2-1, prp8-1, or prp5-1 all exhibit conditional growth phenotypes, showing nearly wild-type growth at 25 \u00b0C but an inability to support growth at 37 \u00b0C. A comparison of growth at intermediate temperatures allows these mutations to be ordered according to the strength of the defect they impart: prp2-1 is the most severe defect as it is unable to support growth above 25 \u00b0C; prp8-1 is slightly less severe as it can support weak growth at 30 \u00b0C; and prp5-1 is the weakest as it shows nearly wild-type growth even at 33 \u00b0C. As seen in As expected, inactivation of each of these factors results in inhibition of splicing of a large number of intron-containing transcripts. Interestingly, while the overlapping set of transcripts that are affected by inactivation of all three factors is quite large, a closer examination suggested important differences in the particular transcripts whose splicing was affected and prompted us to more carefully compare the profiles of these mutants. Microarray experiments were performed that directly compared the pairwise behaviors of these mutant strains C, revealprp2-1 strain than in either the prp8-1 or prp5-1 strains, and a larger accumulation in the prp8-1 strain than in the prp5-1 strain. Interestingly, the molecular splicing defects observed for this class of transcripts correlate with the conditional growth defects: the prp2-1 mutation produces the strongest phenotype, followed by the prp8-1 mutation, and finally the prp5-1 mutation. Notably, as indicated in The second class is also composed of transcripts that exhibit a canonical splicing defect in response to inactivation of each of the single mutants, but for whom the magnitude of the defect is different for each of the mutants. About 100 transcripts behave in this fashion, exemplified by those indicated with a green bar in prp2-1 mutation, but shows little defect in response to either the prp8-1 or prp5-1 mutations. Conversely, the splicing of the Rpl19b transcript is affected by both the prp8-1 and prp5-1 mutations, but is largely unaffected by the prp2-1 mutation. Likewise, the Hnt1 transcript shows little defect in response to the prp8-1 mutation, while splicing of the Cox4 transcript is strongly affected by the prp5-1 mutation but is only mildly affected in the prp2-1 strain, the inverse of the ordering seen for the second class of transcripts described above.The final class is composed of a smaller number of transcripts, those that exhibit splicing inhibition in response to one or two of the mutant strains but not all three. Examples of several transcripts belonging to this class are shown in Given the predominance of RPG transcripts in the second class of transcripts, we sought to identify properties of these transcripts that might distinguish them from the others. As an initial attempt to identify such differences, we asked whether RPG transcripts showed a difference in splicing efficiency relative to other transcripts under standard laboratory growth conditions in wild-type cells. Quantitative RT-PCR was performed using primers specific to both intron and exon regions of 12 different intron-containing genes. Using genomic DNA to generate standard curves, relative copy numbers of both the precursor and total mRNA species were then calculated and compared. As seen in prp8-1 mutation or the temperature-sensitive prp8-101 mutation, respectively, to the nonpermissive temperature and compared to similarly treated wild-type strains. Strikingly, the splicing of most intron-containing transcripts is almost completely unaffected by the prp8-101 mutation. Instead, this mutation appears to affect the splicing of only a small number of transcripts. As it had previously been shown that the prp8-101 mutation results in a defect in catalyzing the second transesterification reaction [prp16-302 mutation to the nonpermissive temperature of 16 \u00b0C. The broad splicing defect seen for this mutant suggests that the differences between the prp8-1 and prp8-101 splicing profiles are not simply the result of comparing mutants defective for the first or second chemical steps, but rather truly reflect allele-specific differences in substrate specificity.The differences that we observed between mutations in different spliceosomal factors led us to ask whether distinct mutations in a single factor might also cause such transcript-specific responses. Shown in reaction , we examreaction . Figure brr5-1 mutant is quite similar to that seen for many other canonical splicing mutants. While this mutant was originally isolated as a splicing mutant [rna14-64 mutant, which is also defective in 3\u2032 end formation [The transcript-specific phenotypes that we observed with this small subset of conditional mutants compelled us to examine the effects of a much wider variety of spliceosomal defects. To facilitate these experiments, a set of high-throughput methods was developed that allowed us to automate many of the steps in a microarray experiment see . Using tg mutant , it has g mutant ,23. By cormation , produceprp19-1 mutation does affect the splicing of many pre-mRNAs, it has little effect on the splicing of this transcript. A comparison of the two U3 paralogs, SNR17A and SNR17B, whose mature products are nearly identical but whose introns share little sequence homology, also shows some differences in their responses to some of the splicing mutants. For example, the U3a transcript shows a stronger defect in response to the prp5-1 mutation than the U3b transcript.Importantly, a careful examination of the response of individual transcripts to this panel of mutations makes clear the fact that all transcripts are not equally affected by mutations in core components. To exemplify this point, RPS30 paralogs. Whereas splicing of the Rps30b transcript is affected by most of the splicing mutants examined, the Rps30a transcript is not. The splicing of the Rps30a transcript shows a strong defect in response to the prp2-1 and prp16-302 mutations, a somewhat weaker response to the prp28-1, brr2-1, brr1-1, and brr5-1 mutations, and very little defect in most of the other mutants. The RPL19 paralogs also display divergent behaviors: whereas the Rpl19a transcript shows a similar defect in response to each of the mutants, the Rpl19b transcript shows strong defects in response to the prp8-1, prp5-1, and prp4-1 mutants, but less severe defects in response to the other mutants. Differences can also be seen between non-RPG transcripts by comparing the behavior of the Act1 and Tef4 transcripts, for example.For some transcripts, the subset of mutations that affect their splicing is much different. A dramatic example of this is provided by the S. cerevisiae to mutations in core spliceosomal factors. For largely technical reasons, most experiments designed to study the efficiency of pre-mRNA splicing to date have focused on a relatively small number of transcripts, leaving open the question of how much variety there is among different spliceosomal substrates. The experiments presented here overcome this limitation by utilizing splicing-specific microarrays that allow for the simultaneous examination of all spliceosomal substrates. Splicing-specific microarrays are particularly amenable to studies in budding yeast, where there are a limited number of intron-containing genes and there is very little alternative splicing. By using this approach to examine the time-resolved effects of inactivating 23 different factors involved in pre-mRNA processing, the work presented here reveals a complex relationship between the activity of the core spliceosome and the full complement of transcripts with which it must interact.Here we describe the results of genome-wide experiments designed to examine the in vivo responses of all intron-containing transcripts in PRP2, PRP8, and PRP5 shown in prp2-1, prp8-1, and prp5-1 mutations, the strengths of the splicing defects imparted by these mutations are variable. Whereas the splicing of many transcripts encoding factors other than ribosomal proteins is equally inhibited by mutations in any of these three factors, the strength of the defect observed in the splicing of most ribosomal protein gene transcripts is dependent upon the particular defect in the spliceosome. For these mutations, the strength of the molecular splicing defect correlates with the temperature sensitivity that the mutation imparts upon the strain: prp2-1 causes the strongest defect and prp5-1 causes the weakest. While the fraction of new transcript from any single gene whose splicing is inhibited cannot be determined from these experiments alone, these results nevertheless demonstrate that a greater fraction of most RPG transcripts is blocked by the prp2-1 mutation than by either the prp8-1 or prp5-1 mutations. This difference is unlikely to simply reflect differences in the severity or onset of the protein mutations because nearly identical fractions of newly transcribed Act1, for example, are blocked by all three of the spliceosomal mutations. Rather, it suggests that the RPG-encoding transcripts are fundamentally different from the other transcripts in their susceptibility to these mutations.A large collection of data such as this offers a unique opportunity to both compare and contrast the behaviors of all 250 intron-containing transcripts according to their individual responses to the different spliceosomal mutations. From this perspective, the detailed comparison of mutations in We have not yet been able to identify any single feature of intron-containing RPGs that explains this altered susceptibility. While it is true that RPG introns tend to be longer than non-RPG introns, and that RPGs tend to be among the more highly transcribed genes in the genome, neither of these features alone seems sufficient to explain the different responses. The Act1 transcript, for example, is interrupted by an intron that is similar in length (308 nucleotides) to an average RPG intron (mean length of 402 nucleotides), and is transcribed at a similar rate as many RPGs , yet itsIn an effort to identify properties that might differentiate RPG transcripts from other intron-containing transcripts, we used quantitative RT-PCR to determine the amount of precursor and mature mRNA species present in wild-type cells for a variety of different intron-containing genes. While all 12 of the transcripts that we examined were efficiently spliced (>80%), the RPG transcripts showed remarkably high partitioning toward mature species. It is noteworthy that, in spite of the high levels of total transcript present for most RPGs, the amount of precursor species present is remarkably low relative to the other intron-containing transcripts. For example, while there is more than ten times as much total Rps21b transcript in a cell as there is total Nmd2, 50 times less Rps21b pre-mRNA can be detected as compared to Nmd2 pre-mRNA.It is tempting to speculate that the different splicing efficiencies and the different susceptibilities to spliceosomal mutations displayed by the RPGs may be mechanistically related. For example, while much recent progress has been made in identifying protein components of the spliceosome , it remaprp2-1, prp8-1, and prp5-1 mutations, or the entire panel of factors as shown in RPS30 paralogs offer the most dramatic example of these differences. Whereas splicing of the Rps30b transcript is adversely affected by almost all of the spliceosomal mutations that we examined, splicing of the Rps30a transcript is only affected by a small subset of these mutants. The particular features unique to each of these transcripts that underlie this differentiation remain unclear. Presumably the ability of the Rps30a transcript, for example, to be efficiently spliced in the prp8-1 mutant does not indicate that Prp8 function is unnecessary for the splicing of this transcript. Rather, it suggests that the unique set of interactions that this transcript forms with the spliceosome are less sensitive to the defect imparted by this particular mutation in Prp8. Importantly, because the Rps30a and Rps30b transcripts behave so differently in response to so many mutations, they should prove to be extremely useful as tools for more traditional biochemical experiments designed to understand the function of individual spliceosomal factors.Importantly, not all RPG transcripts behave similarly in these experiments. Whether considering the simple comparisons of the prp8-101 mutation, which appears to represent such a mutant, knowing the identity of transcripts that are either strongly affected or unaffected provides important information for designing more traditional biochemical experiments in the future to examine the mechanism of its activity in the spliceosome. Likewise, the genome-wide splicing profiles derived from similar mutations in other factors might prove more revealing about the mechanistic role of those factors.Most of the mutants that we have examined here were isolated from conditional lethal screens. As such, this subset of mutations may be biased towards those with strong growth defects and hence broad transcript specificity. Perhaps because of this, we have found it difficult to associate a particular mechanistic role with any of these factors based simply on the subset of pre-mRNAs whose splicing is affected in these experiments. Rather, our findings seem to indicate that the signature of introns that are affected by a particular spliceosomal mutation is unique to that mutation. Nevertheless, it is tempting to imagine isolating other mutations in these core components that affect the splicing of a smaller subset of transcripts. Such mutations may be more difficult to isolate, as they may be aphenotypic for growth under standard laboratory conditions. Nevertheless, a genome-wide splicing analysis of such a mutant may provide important insights into the activity of the protein. Indeed, for the Drosophila melanogaster show differential effects on splicing activity at alternative splice sites [prp8-101 mutation, the mutations associated with retinitis pigmentosa may cause defects in the splicing of a distinct group of transcripts, presumably uniquely associated with retinal function.Interestingly, the capacity of core spliceosomal components to elicit transcript-specific changes in splicing activity does not appear to be limited to yeast. Recent RNAi-mediated depletion experiments targeting core spliceosomal components in ce sites . Given tce sites \u201328. As sS. cerevisiae lacks the large SR and hnRNP protein families responsible for modulating transcript-specific splicing activity in higher eukaryotes, the observations presented in the current work suggest that the core spliceosomal components themselves could be targets of environmental regulation. As seen with stable genetic modifications here, post-translational modifications of these components might similarly facilitate transcript-specific regulation of pre-mRNA splicing. Such modifications would allow the cell to both rapidly and specifically regulate the production of translatable mRNA for particular transcripts in a reversible manner. Whether or not the complex relationships between transcripts and core spliceosomal components revealed by these mutant studies represent features of a system that has evolved to be the target of biological regulation is a provocative question that remains to be fully addressed.We have recently shown that the yeast spliceosome can rapidly and specifically alter the splicing efficiency of distinct subsets of transcripts in response to at least two unrelated environmental stresses . Notably, in response to amino acid starvation, the splicing of virtually all RPG transcripts is specifically downregulated. Given that cis features of the intron, nor does the biochemical activity presumed to be affected by a mutation appear to dictate which introns will be affected. Instead, in much the same way that transcription is now known to be regulated by the combinatorial control of its holoenzyme, it appears that the efficiency of pre-mRNA splicing may be dependent upon the complexes present in the spliceosome as well as the particular transcript upon which it assembles.The work presented here does however suggest that the relationship between the spliceosome and the full complement of transcripts with which it must interact is much more complex than previously believed. Important questions for the future concern both the mechanism by which the spliceosome distinguishes among these substrates and the biological rationale for this specificity. The simple hypothesis that all mutations in splicing factors that inhibit the growth of a yeast cell would also inhibit the splicing of all (expressed) intron-containing transcripts is inconsistent with our observations. The splicing efficiency of a given intron can not yet be ascribed to obvious 600 = 0.5 and A600 = 0.7. An initial 15 ml sample was collected at 25 \u00b0C prior to initiation of the time course. Cells were collected by filtration using Millipore HAWP0025 filters (http://www.millipore.com). The filters were immediately frozen in N2(l). The culture flasks were then transferred into water baths at either 16 \u00b0C or 37 \u00b0C, with additional 15 ml aliquots removed and collected at the appropriate times. Total cellular RNA was isolated as previously described [http://www.eppendorf.com) were used during phase separation steps. Also, RNA samples were precipitated using isopropanol.Unless otherwise indicated, cultures were grown according to standard techniques in rich medium supplemented with 2% glucose at 25 \u00b0C. Both mutant strains and the corresponding wild-type strain were grown in parallel, 100 ml cultures until their optical densities were between Aescribed  with a f2, 10 mM DTT, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 0.3 mM TTP, 0.2 mM aa-dUTP, 12.5 \u03bcg dN9 primer, and 5 ng murine Moloney leukemia virus (M-MLV) RT. Primers were annealed to the RNA by heating to 65 \u00b0C for 5 min in the presence of buffer and salt alone. Reactions were allowed to incubate at 42 \u00b0C for at least 2 h. Remaining RNA was then hydrolyzed by incubation for 15 min at 65 \u00b0C in the presence of 0.1 M NaOH and 10 mM EDTA. This was neutralized with HCl, then purified using a Zymo25 DNA purification column according to the manufacturer's protocol (http://www.zymoresearch.com). Typical yields ranged from 10 to 15 \u03bcg of cDNA from 25 \u03bcg of starting material. The purified cDNA was then conjugated to the appropriate fluorescent dye in a 10 \u03bcl reaction containing 50 mM sodium bicarbonate (pH 9.0), 50% DMSO, and \u223c20 \u03bcg of NHS-derivatized fluorophore. Reactions were incubated in the dark at 60 \u00b0C for 60 min, after which time the cDNA was repurified using a Zymo25 DNA purification column.For each microarray sample, cDNA was prepared from 25 \u03bcg of total RNA in a 50 \u03bcl reaction mixture containing 50 mM TrisHCl (pH 8.3), 75 mM KCl, 3 mM MgCl600 = 0.5 and A600 = 0.7. After the strains were synchronized, the cultures were transferred into the wells of a 96-well growth plate at 25 \u00b0C. A reverse time course was then initiated using a multichannel pipettor at the appropriate times to transfer the cultures into a second 96-well plate submerged in a 37 \u00b0C water bath (g for 5 min. The cell pellets were then frozen in N2(l). With the help of a Biomek FX liquid handling system , total cellular RNA was then isolated using largely standard procedures. Typical yields from a single 1.8 ml well ranged from 15 \u03bcg to 25 \u03bcg of total RNA. For each well, cDNA synthesis was performed in a 50 \u03bcl reaction as described above. After cDNA synthesis, the remaining RNA was hydrolyzed by addition of 25 \u03bcl of 0.3 M NaOH/0.03 M EDTA and heating to 60 \u00b0C for 15 min. This solution was neutralized by addition of 25 \u03bcL of 0.3 M HCl. To purify the cDNA from this solution, 0.5 ml of cDNA binding buffer  was added to each well. This material was passed over a 96-well glass-fiber filtration plate . The filters were then washed once with 0.5 ml of Wash I , and once with 0.5 ml of Wash II (80% EtOH). Purified cDNA was then eluted in two steps of 100 \u03bcl water. The appropriate dyes were then coupled to the cDNA as described above. Fluorescently labeled cDNA was then repurified using 96-well glass-fiber filtration plates as described above.Cultures were grown in sets of four mutant strains along with their corresponding wild-type strains in rich medium in flasks at 25 \u00b0C until their optical densities were between Ater bath . The enthttp://www.fermentas.com) according to the manufacturer's protocol. The cDNA was then produced from 2 \u03bcg of total RNA in a 20 \u03bcl reaction mixture containing 50 mM TrisHCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dNTP, 25 nM each gene-specific primer, 2.5 \u03bcg dN9 primer, and 5 ng M-MLV RT. Quantitative PCR was then performed using an Opticon from MJ Research (http://www.mjresearch.com). Primer sets used in this study are shown in 6 molecules was used to generate the standard curves. Genomic DNA was purified using a ZR Fungal DNA kit (Zymo Research) according to the manufacturer's protocol. The copy numbers presented in the Table have been normalized to the least abundant species detected in these experiments, the precursor isoform of the RPS21b transcript. Importantly, these copy numbers do not represent cellular copy numbers.Total cellular RNA for quantitative RT-PCR experiments was prepared as described above. Prior to conversion to cDNA, the RNA was treated with DNase I  and premature mRNA feature probes (intron oligos) 32 nucleotides in length were designed using a previously described algorithm . Intron http://www.agilent.com) at 60 \u00b0C for a period ranging from 12 to 16 h.Probes were printed on poly-lysine-coated glass slides using standard techniques. Each probe was print on the array three times by each of two different printing pins . Image analysis was performed using Axon Instruments GenePix Pro version 4.0 . Ratio values derived from the median pixel intensities for the 635 nm and 532 nm images of each spot were used to represent probe behaviors in data preprocessing analysis. A standardized qualitative assessment of array quality was performed using the Bioconductor arrayQuality package  [2 transformed and normalized within each array using global loess regression implemented in the Bioconductor marray package [Microarray images were acquired using an Axon Instruments GenePix 4000B scanner, reading at wavelengths of 635nm and 532nm  ,32. Arra package . All exp package . Linear For downstream hierarchical clustering, feature ratio values were calculated by averaging the median within-array spot replicate measures between replicate arrays. Where appropriate, replicate variation was used to weight feature measurements. Hierarchical clustering was performed using the C Clustering Library version 1.32 . Data wehttp://www.ncbi.nlm.nih.gov/geo).All microarray data are available at the Gene Expression Omnibus (GEO) repository at the National Center for Biotechnology Information Click here for additional data file.Figure S2prp2-1 (blue), prp8-1 (red), or prp5-1 (green) samples. Input material was normalized using the average value obtained from probes targeting both NRG1 and ECO1 transcripts. Values shown are derived from triplicate measurements with error bars included.(A) Quantitative RT-PCR (QPCR) experiments comparing wild-type samples with (B) Comparison of QPCR and microarray values for genes shown in (A).(1.4 MB EPS)Click here for additional data file.Figure S3Ratio values corresponding to the precursor species of the indicated genes (top) derived from each of three mutant analyses are plotted against initial precursor values as determined in (1.3 MB EPS)Click here for additional data file.Figure S4Flow diagram of splicing and spliceosomal recycling pathways showing putative stage of action of each interrogated allele. Alleles in factors involved in 3\u2032 end processing and mRNA nuclear export were also tested in this study.(1.2 MB EPS)Click here for additional data file.Figure S5(2.6 MB EPS)Click here for additional data file.Figure S6(A) Experimental time courses of temperature shifts were performed in a 96-well format. Samples were from transferred from flasks at 25 \u00b0C to a 96-well plate shaking in a 37 \u00b0C bath in a reverse time course. Matched wild-type and mutant samples were collected in the same 96-well plate to ensure consistency in time points between hybridized samples . Samples were collected for two biological dye-flipped array replicates in each 96-well plate .600 = 0.5 and A600 = 0.7 at 30 \u00b0C purified using either the standard or the high-throughput method (see (B) Replicate samples of total RNA from wild-type cells grown to optical densities between Athod see  are show(C) RT efficiency of total RNA samples shown in (B).(4.2 MB EPS)Click here for additional data file.Figure S7To design mature mRNA probes see , probe M(1.2 MB PDF)Click here for additional data file.Figure S8Each biological sample is analyzed with three layers of data replication. Each probe is printed on the arrays three times by each of two different printing pins. Within a block (printed by a single printing pin) the three spot replicates are printed in the same column, separated vertically by eight rows. This vertical separation allows for assessment of top-to-bottom spatial spot ratio and intensity bias within each block. Block replicates (printed by two different pins) are oriented on the array with 180\u00b0 rotational symmetry . Horizontal and vertical separation of blocks allows for between-block assessment of both top-to-bottom and left-to-right spatial spot ratio and intensity bias. Finally, each experiment is assayed on two dye-flipped biological replicate arrays.(7.0 MB PDF)Click here for additional data file.Figure S9prp8-1 versus wild-type 30-min 37 \u00b0C temperature shift.An example of a standard qualitative assessment of array quality, with array data from the 2 ratios  compared to total spot intensities , with traces showing the average behavior of each of the 16 blocks corresponding to individual printing pins.(A) Prenormalization spot log(B) Spatial visualization of prenormalization M-value ranks across the array.(C) Hexagon binning plot showing distribution and density of global Loess normalized M-values versus A-values.(D) Spatial visualization of global Loess normalized M-values across the array.(2.3 MB EPS)Click here for additional data file.Figure S102 ratios  for every probe on the prp8-1 versus wild-type 30-min 37 \u00b0C temperature shift array. Each set of six replicate probes is plotted on a single line vertically. M-values for probes printed in the first set of printing blocks (blocks 1\u20138) are plotted in blue, while M-values for probes printed in the second set of printing blocks (blocks 9\u201316) are plotted in orange. Spot replicate sets are arranged vertically based on the median M-value of each set.(A) Dotplot of normalized, unfiltered, log(B) Density trace (Gaussian smoothing) of standard deviations for each replicate set on the array shown in (A). Three different density traces are shown for the three different types of probe sets.prp8-1 versus wild-type 30-min 37 \u00b0C temperature shift array (Exp/Ref) and the matched dye-flipped replicate array (Ref/Exp). Points corresponding to total mRNA features, pre-mRNA features, and mature mRNA features are plotted in blue, red, and green, respectively. Legend shows Pearson correlations between the values for each of these types of features in the two arrays.(C) Plot of normalized M-values on the (2.6 MB EPS)Click here for additional data file.Table S1(14 KB XLS)Click here for additional data file.Table S2prp2-1 mutation versus wild-type cells to 37 \u00b0C for 30 min. The \u201cORF Name\u201d column lists the standard yeast ORF name associated with each intron. M values represent averaged, log2-transformed ratios. The B-statistic represents the log posterior odds ratio.Evidence for differential expression of intron sequences after shifting cells carrying the temperature sensitive (46 KB XLS)Click here for additional data file.Table S3prp8-1 mutation versus wild-type cells to 37 \u00b0C for 30 min. The \u201cORF Name\u201d column lists the standard yeast ORF name associated with each intron. M values represent averaged, log2-transformed ratios. The B-statistic represents the log posterior odds ratio.Evidence for differential expression of intron sequences after shifting cells carrying the temperature sensitive (46 KB XLS)Click here for additional data file.Table S4prp5-1 mutation versus wild-type cells to 37 \u00b0C for 30 minutes. The \u201cORF Name\u201d column lists the standard yeast ORF name associated with each intron. M values represent averaged, log2-transformed ratios. The B-statistic represents the log posterior odds ratio.Evidence for differential expression of intron sequences after shifting cells carrying the temperature sensitive (46 KB XLS)Click here for additional data file.Table S5prp8-101 mutation versus wild-type cells to 37\u00b0 C for 30 min. The \u201cORF Name\u201d column lists the standard yeast ORF name associated with each intron. M values represent averaged, log2-transformed ratios. The B-statistic represents the log posterior odds ratio.Evidence for differential expression of intron sequences after shifting cells carrying the temperature sensitive (46 KB XLS)Click here for additional data file."}
+{"text": "Anopheles gambiae sensu stricto (A. gambiae), provides a unique opportunity to study the evolution of reproductive isolation because it is divided into two sympatric, partially isolated subtaxa known as M form and S form. With the annotated genome of this species now available, high-throughput techniques can be applied to locate and characterize the genomic regions contributing to reproductive isolation. In order to quantify patterns of differentiation within A. gambiae, we hybridized population samples of genomic DNA from each form to Affymetrix GeneChip microarrays. We found that three regions, together encompassing less than 2.8 Mb, are the only locations where the M and S forms are significantly differentiated. Two of these regions are adjacent to centromeres, on Chromosomes 2L and X, and contain 50 and 12 predicted genes, respectively. Sequenced loci in these regions contain fixed differences between forms and no shared polymorphisms, while no fixed differences were found at nearby control loci. The third region, on Chromosome 2R, contains only five predicted genes; fixed differences in this region were also verified by direct sequencing. These \u201cspeciation islands\u201d remain differentiated despite considerable gene flow, and are therefore expected to contain the genes responsible for reproductive isolation. Much effort has recently been applied to locating the genes and genetic changes responsible for reproductive isolation between species. Though much can be inferred about speciation by studying taxa that have diverged for millions of years, studying differentiation between taxa that are in the early stages of isolation will lead to a clearer view of the number and size of regions involved in the genetics of speciation. Despite appreciable levels of gene flow between the M and S forms of A. gambiae, we were able to isolate three small regions of differentiation where genes responsible for ecological and behavioral isolation are likely to be located. We expect reproductive isolation to be due to changes at a small number of loci, as these regions together contain only 67 predicted genes. Concentrating future mapping experiments on these regions should reveal the genes responsible for reproductive isolation between forms.The African malaria mosquito,  Using DNA microarrays, the authors identify 3 small regions of the genome that differ between two forms of hybridizing mosquitoes; regions that are likely to contain the genes responsible for reproductive isolation. Uncovering the genetic basis for reproductive isolation is a key to understanding how biological diversity is generated. Many researchers have used quantitative trait locus (QTL) mapping experiments to find the number and size of regions involved in both pre- and post-mating isolation between species e.g., \u20136). Alth. Alth6])Anopheles gambiae sensu stricto (A. gambiae), is the type species of the Anopheles gambiae sensu lato complex: a group of seven closely related African species morphologically indistinguishable as adults [A. gambiae is further subdivided into two partially isolated taxa known as the M form and S form [s adults  and incos adults ,16. The s adults . In addid S form . These fd S form . Subsequd S form ,21. Whend S form , suggestd S form ), but thd S form \u201327.M and S forms of A. gambiae and to delineate the number and size of regions that do not introgress\u2014which may contain genes involved in reproductive isolation\u2014we hybridized DNA of single mosquitoes from population samples of M and S forms to Affymetrix GeneChip microarrays. Recent studies in Saccharomyces cerevisiae [Arabidopsis thaliana [Arabidopsis thaliana inbred strains and their recombinant inbred line, and were able to precisely delineate which regions of the recombinant line came from either parental line. Our goals were (1) to locate regions of the genome where M and S forms differed; (2) to test whether the observed pattern of differentiation resulted from selection against gene flow or could be explained by genetic drift or other processes; and (3) to determine what this genomic pattern could tell us about speciation and adaptive radiation.To better examine the genetic basis for the maintenance of reproductive isolation between the revisiae  and Arabthaliana  have shothaliana  used thiM form and seven samples of S form mosquitoes from areas of Cameroon where they are sympatric (see A. gambiae [M and S forms possess the same (standard) karyotype [We used seven samples of tric see , and whe gambiae ,30. In Caryotype .Plasmodium/Anopheles GeneChip array to the most recent A. gambiae genome assembly and removed probes with multiple exact matches, which generated a marker map of 142,065 unique probes .We remapped each 25-bp probe on the Affymetrix obes see . Whole gt-test p-values for each probe and considered probes with p < 0.01 to be candidate single-feature polymorphisms . The null hypothesis in this analysis, a random distribution of SFPs, could be violated simply because of linkage disequilibrium of probes within a gene. We permuted probesets\u2014preserving the association of probes within a gene\u2014to test for this effect , but the 2R regions were not significant. For the 2R region detected in both analyses, nonsignificance may be due simply to its small size in relation to the size of sliding windows: seven of 11 SFPs in this window fell within four probesets, spanning only 40 kilobases (kb). Overall, the significance of the differentiated regions on Chromosomes 2L and X is strongly supported by all analyses, and the small region on 2R is suggestive: these three regions are our candidate \u201cspeciation islands.\u201d Using the HMM, we estimated the sizes of these regions to be 2,160 kb, 566 kb, and 37 kb for Chromosomes 2L, X, and 2R, respectively. We expect these values to be underestimates for the regions on 2L and X because some heterochromatic portions of the neighboring centromeres have not been assembled. The number of predicted genes in each chromosomal region is 50 in 2L, 12 in X, and five in 2R.Differentiation between forms is shown in s . We alsfect see . After cfect see . The 2L p = 0.00005; Chromosome X: five fixed, none shared within the differentiated region, none fixed, four shared outside the differentiated region, Fisher's exact test, p = 0.008). The intron of the P450\u20132 gene, in the divergent Chromosome X region, also had a 51-bp indel fixed between forms. The level of polymorphism within differentiated regions on Chromosomes 2L and X was low within each form , as would be expected if these regions had low rates of recombination because of proximity to centromeres. The nearby control region on X also had low polymorphism  has no known function, and a BLASTn search yielded significant similarity only to an adjacent gene in A. gambiae that is also within the differentiated region but was not sequenced in our study. The two other genes within the island had no fixed differences, but all three loci had highly unequal levels of diversity between forms; S form mosquitoes showed up to 20 times the levels of nucleotide polymorphism as M form individuals. Gene UNK1 showed the greatest asymmetry in polymorphism, with 21 SNPs in the S form sample and two in the M form sample. There is no evidence from tests of selection [On Chromosome 2R we sequenced five loci: three loci inside the 37-kb region that was detected in both of our whole-genome analyses and two control loci adjacent to this region . We simulated two types of loci: one with levels of nucleotide polymorphism and recombination typical of most of our control regions, and one with 10-fold reductions in effective population size and recombination rate typical of most of our differentiated regions (see p = 0.0032). Conservatively considering the two sequenced genes within each of our Chromosome 2L and Chromosome X islands as single loci, the probability of sequencing loci from both regions and observing fixed differences without shared polymorphisms\u2014even with a much reduced effective population size\u2014is p < 0.0001. It is therefore unlikely that decreased variability alone is responsible for the observed differences between forms.Additional control sequences from Chromosome 3R showed shared polymorphisms between forms and no fixed differences, despite low levels of variability at some loci . This hi density . The lac density  to gener density ). Estima density   [M/S genotypes have been found (1.1% of larvae and 0.3% of adults in a population where M and S are sympatric) [M and S forms in Cameroon found stF values between forms that were consistent with substantial migration rates . Microsatellite stF values are currently being calculated for the mosquitoes used in our study, with comparable results .Our genome-wide array analysis and our analysis of sequence polymorphism clearly show that differentiation between the matings) ,21, and mpatric) . A previorted in  yields 1In contrast to the low levels of differentiation found throughout most of the genome, our array experiments revealed three small regions to be significantly differentiated between forms. Sequences from islands on Chromosomes 2L and X contain 13 fixed differences and no shared polymorphisms. One gene within the third island, a 37-kb region on Chromosome 2R, also shows only fixed differences between forms.p < 0.0001) suggests that differentiation in these regions is due to selection against hybrid genotypes during backcrossing. This conclusion supports the prediction that when gene flow is present, differentiation between incipient species can be limited to small regions surrounding isolating genes [In the early stages of divergence, above-average differentiation is expected between regions of low recombination. We conducted coalescent simulations to test whether the observed differentiation on Chromosomes 2L and X could plausibly result from neutral scenarios. Rejection of this neutral hypothesis , Mutanguene , and Tiko , in Cameroon in 2003 by the lab of G. C. Lanzaro , who graciously shared samples for this study. DNA was extracted following Post et al. [A. gambiae from A. arabiensis [A. gambiae M and S forms [g for 10 min, and the pelletized DNA was dried and resuspended in 50 \u03bcl of ddI H2O. Microarrays were hybridized by the University of California at Davis School of Medicine Microarray Core Facility using genomic DNA in place of cDNA in the standard Affymetrix protocol .Mosquitoes were collected in the towns of Buea . Each probe on the array was blasted against the most recent A. gambiae genome assembly in order to remove probes with more than one exact match and to remap all probes onto the current assembly. We considered probes with two tailed t-test p-values less than 0.01 to contain SNPs between forms . These analyses were carried out on each chromosome individually; the average window size was approximately 500 kb, with a range of approximately 60\u20132,000 kb, depending on the density of probes in each region. Significant probes could be more clustered than random because of the shared history of linked probes, so the sliding window analysis was repeated on 1,000 permuted datasets to test the effect of short-range linkage disequilibrium on our conclusions. For each permutation, probesets were reshuffled, but probes within a probeset remained associated. The 300-probe window with the highest number of significant probes in each permutation was recorded, and significance was assigned based on the number of permutations containing a window with differentiation equal to the observed value. As an additional test, we constructed a HMM [p < 0.01 counted as one observation). Nonindependence could also arise because of deletions covering whole probesets; deletions were characterized by low p-values throughout a probeset, and were collapsed into one observation for the analysis. The data shown in Raw hybridization intensities were normalized in R via RMA  using the method ,29, and ed a HMM  to segmehttp://www.codoncode.com, which uses ABI quality scores and Phred/Phrap to call bases, find heterozygous SNPs, and correct for heterozygous indels. Analysis of polymorphism and divergence was done in DNAsp (http://www.ub.es/dnasp/). The regions we sequenced covered nine probes with uncorrected p-values less than 0.01. The expected nucleotide difference was found in seven probes (78%), and one of the remaining two probes overlapped with a probe that contained the expected difference, highlighting the nonindependence of overlapping probes . Because our samples were potentially heterozygous, we expected probes with low p-values to be either fixed differences or nucleotides with highly differentiated frequencies between forms. Sequencing of these six probes verified this expectation, as the detected nucleotide difference was either fixed between forms, or nearly fixed with either one or two heterozygous individuals for the rare allele.All products were sequenced in both directions; see S = 27) and the differentiated loci on the 2L loci within the island (S = 12). We estimated migration to be 4emN = 10 for the control loci and 4emN = 1 for the differentiated loci [r = 1 \u00d7 10\u22128 for recombination per site in control regions  and r = 1 \u00d7 10\u20139 for recombination per site in differentiated regions . All simulations were run 10,000 times, where the output of MS was parsed to count the number of fixed and shared polymorphisms for each run. To obtain the p-value associated with observing two loci with fixed differences and no shared polymorphisms, we sampled two loci for each of the 10,000 iterations and counted the number of times both showed this pattern.Using the program MS , we geneted loci . BecauseTable S1(23 KB XLS).Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the gene sequences discussed in this paper areAY825543\u2013AY825922 and DQ080663\u2013DQ080909.The GenBank ("}
+{"text": "Anopheles gambiae gene, ENSANGG00000017398. Based on phylogenetic analysis, this gene belongs to the same lineage as Heat shock protein cognate 70-4 (Hsc70-4) in Drosophila. Accordingly, we propose to name this gene Heat shock protein cognate 70B (HSC70B). We previously reported that expression of HSC70B and other genes including elongation factor-1\u03b1 (EF-1\u03b1) and the agglutinin attachment subunit (agglutinin) were up-regulated in o'nyong-nyong virus (ONNV)-infected female An. gambiae. Double-stranded RNA interferences have been applied to further investigate HSC70B, EF-1\u03b1 and the agglutinin functions in ONNV replication in An. gambiae.Phylogenetic and functional analysis was conducted on an An. gambiae compared to the control mosquitoes that were co-injected with ONNV and dsRNA of \u03b2-galactosidase . ONNV titers from mosquitoes co-injected with dsHSC70B were about 9-fold higher at 6 days post-injection (d.p.i.) as compared to the control mosquitoes. By using ONNV tagged with enhanced green fluorescent protein (ONNV-eGFP), co-injection of ONNV-eGFP with dsHSC70B also showed approximately 2 ~ 3-fold higher GFP expression rates than the controls in the head, thorax, and abdomen of the mosquito. Furthermore, co-injection of ONNV with dsHSC70B significantly reduced the lifespan of adult mosquitoes as compared with the control, co-injection of ONNV with ds\u03b2-gal treated mosquitoes.Among these three RNAi silenced genes, only dsRNAs of HSC70B (dsHSC70B) promoted ONNV replication in adult An. gambiae. Biological implications of these findings are that while mosquitoes allow ONNV to replicate in them, they also check viral titers so that ONNV infection will result in no harmful effect on mosquitoes. Therefore, mosquitoes can function as vectors of ONNV transmission to humans while ONNV infection in An. gambiae remains asymptomatic.These results indicate that HSC70B plays important roles in homeostasis and suppression of ONNV replication in the vector,  Anopheles gambiae and An. funestus [The arbovirus, o'nyong-nyong virus (ONNV) belongs to genus Alphavirus, and is an enveloped, single stranded, (+) RNA virus with a genome of approximately 12 kb [funestus . ONNV wafunestus . Recentlfunestus ,6.An. gambiae genome has allowed us to investigate modulation of mosquito gene expression resulting from arbovirus infection. Genome-wide screening of differentially expressed transcripts of ONNV-infected female An. gambiae relative to na\u00efve females was conducted at 14 day p.i. [An. gambiae compared with controls by cDNA microarrays followed by paired t-test and quantitative real time PCR (qRT-PCR) analysis. The products of the seven genes are seemingly involved in protein translation, DNA replication, or intracellular transport pathways [Although mosquitoes are critical vectors in many arboviral transmission cycles, there is limited information on how arboviruses influence mosquito gene expression and how mosquito immune systems defend arthropod vectors from deleterious consequences of viral infection. The recent completion of the sequencing of the day p.i. . Seven gAnopheles EF-1\u03b1 may have a similar role for ONNV replication in An. gambiae. Lastly, agglutinin is a membrane attachment subunit that may interact with ONNV on the membranes of endosomes and lysosomes. Because non-structural proteins and RNAs of alphaviruses including ONNV are associated with the membranes of modified endosomes and lysosomes in the replication complex [We hypothesize that elevated gene expression of HSC70B for example, may protect the mosquito cells from ONNV-induced molecular damage . Since m complex , agglutiAn. gambiae. The result showed that silencing the HSC70B transcript caused significant increase of ONNV titers in female mosquitoes whilst the remaining two genes had no noticeable effects. Herein, we discuss potential antiviral activity of HSC70B in An. gambiae.Based on these assumptions, HSC70B, EF-1\u03b1 and agglutinin genes were subjected to a detailed functional analysis for their potential involvement in ONNV replication. Using RNAi, we post-transcriptionally silenced target transcripts of the three genes, HSC70B, EF-1\u03b1 and agglutinin by co-injecting dsRNAs of each target transcript with ONNV into female An. gambiae and D. melanogaster indicate that Anopheles HSC70B gene is evolutionarily more conserved with Drosophila Hsc70-1 and Hsc70-4 genes than other Anopheles homologues  at 6 d.p.i.  of mosquitoes receiving ds\u03b2-gal expressed eGFP in thoracic tissues. However, 87% (n = 23) expressed eGFP in thoracic tissues when dsHSC70B was silenced (Table n = 32) and 22% (n = 32) of eGFP expression in head and abdomen, respectively (Table n = 23) and 65% (n = 23) of eGFP expression in head and abdomen, respectively  and S7 (RpS7) were used as internal controls for infection studies of ONNV and the malaria parasite, ectively . When RpAn. gambiae mosquitoes with down-regulated HSC70B alone lead a reduced survival rate (~80%) at 6 days post injection, though it is much less harmful than co-injection of ONNV and dsHSC70B. This suggests that both reduced expression levels of HSC70B gene and increased ONNV infection level synergistically shorten the lifespan of An. gambiae . Stock virus was produced following a single passage in Vero cells maintained at 37\u00b0C in Leibovitz L-15 medium with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 \u03bcg/mL streptomycin. Cell supernatant was harvested when 75% of the cells showed cytopathic effect (3+ CPE). Supernatant containing the virus was collected and titrated. The virus stock contained 2 \u00d7 107 plaque-forming units (pfu)/ml, diluted to 2 \u00d7 106 pfu/ml, aliquoted, and stored at -80\u00b0C.The SG650, strain of ONNV was obtained from the World Reference Center for Arboviruses at the University of Texas Medical Branch, Galveston, TX. Strain SG650 was isolated from human serum in Uganda in 1996  and has AGCAAGGGCGAGGAGCTGTTC-3') Onn-GFP-Pac-R (5'-GACCTTTAATTAATTACTTGTACAGCTCGTCCAT-3'). The PCR product was cloned into AscI and PacI sites of infectious clone pONNic-Foy, provided by K. E. Olson and B. D. Foy , which was previously modified by replacing the T7 promoter with a SP6 promoter. pONNic-Foy clone was derived from pONN.AP3, developed by Brault and others [Not I, which was in vitro transcribed from the SP6 promoter using the mMESSAGE mMACHINE kit  following the manufacturer's instructions. The RNA was electroporated into BHK-21 cells as previously described [The eGFP gene was amplified from pEGFP plasmid  using primers Onn-GFP-Asc-F (5'-GACCTATGGTGd others . Infectiescribed . Cell cuAn. gambiae [6 pfu/ml) were coinjected into the thorax of CO2-anesthetized adult females by using a IM 300 Microinjector . Thus, each mosquito was coinjected with ~3.1 \u00d7 102 pfu of virus and ~625 ng of dsRNA.Templates for the preparation of dsRNA for each candidate gene were PCR-derived fragments flanked by two T7 promoter sequences (TAA TAC GAC TCA CTA TAG) Table . Each PC gambiae  to validEach mosquito was triturated in 1 ml of DMEM, and large particulates were pelleted by centrifugation at 300 r.p.m. and then titrated by standard plaque assay in Vero cells . The plaAn. gambiae. RpS7 gene of An. gambiae was used as an internal control for 23 cycles . To remove genomic DNA contamination, RNA samples were treated with 1.0 \u03bcl DNase I following the manufacturer's instructions . For reverse transcription, 5 \u03bcg of total RNA were reverse transcribed with Superscript III RNase H-reverse transcriptase (Invitrogen). Single-stranded cDNAs of different dilution were amplified by PCR using recombinant Taq DNA polymerase (Invitrogen). To show the RNAi efficiency, primers were made to amplify endogenous agglutinin, EF-1\u03b1 and HSC70b genes of An. gambiae genomic DNA ranging from 116 to 0.0116 ng per reaction. All reactions were performed in triplicate in a total volume of 25 \u03bcl containing 12.5 \u03bcl of SYBR Green PCR Master Mix, 300 nmol of each primer at the following conditions: 50\u00b0C for 2 min, 95\u00b0C for 10 min followed by 50 cycles of denaturation at 95\u00b0C for 15 s, annealing and extension at 60\u00b0C for 1 min. RNA samples were extracted from mosquitoes at 6 days p.i. Sequences of gene-specific primer sets are given in Table qRT-PCR was performed using an ABI 7700 Sequence Detection System . Standard curves were generated for each transcript tested using 10-fold serial dilutions of An. gambiae, 15 females per cohort were intrathoracically co-injected with dsHSC70B and ONNV. For control, 15 females per cohort were intrathoracically coinjected with ds\u03b2-gal and ONNV. Each treated cohort was kept in 8 cm (diameter) \u00d712 cm cages with a cotton wool pad soaked a 10% sucrose solution. The cages were placed at 27\u00b0C and 80% relative humidity under a 12 h light: 12 h dark photoperiod, and mosquito survival was assessed at 24 hours. Survival was defined as the ability of the mosquito to right itself. Experiments for each of the two groups were replicated six times.To evaluate the knockdown effect of HSC70B gene on the survival rate of ONNV infected An. gambiae genome has 10 genes containing the HSP70 domain [Drosophila were obtained from the Berkeley Drosophila Genome Project [The 0 domain . Among t Project ,42.Multiple sequence alignments were performed by using ClustalW v1.81 . PhylogeCS carried out the study with contributions from YSH, TK and DLV. CS drafted the manuscript with contributions from YSH, KT, DLV, SH and FHC. All authors read and approved the final manuscript."}
+{"text": "MicroRNAs are an important class of regulatory RNAs which repress animal genes by preferentially interacting with complementary sequence motifs in the 3' untranslated region (UTR) of target mRNAs. Computational methods have been developed which can successfully predict which microRNA may target which mRNA on a genome-wide scale.We address how predicted target sites may be affected by alternative polyadenylation events changing the 3'UTR sequence. We find that two thirds of targeted genes have alternative 3'UTRs, with 40% of predicted target sites located in alternative UTR segments. We propose three classes based on whether the target sites fall within constitutive and/or alternative UTR segments, and examine the spatial distribution of predicted targets in alternative UTRs. In particular, there is a strong preference for targets to be located in close vicinity of the stop codon and the polyadenylation sites.The transcript diversity seen in non-coding regions, as well as the relative location of miRNA target sites defined by it, has a potentially large impact on gene regulation by miRNAs and should be taken into account when defining, predicting or validating miRNA targets. Recent years have seen an increased appreciation for the importance of post-transcriptional regulation in eukaryotic organisms ,2. The sThe cis-regulatory sequences of many known post-transcriptional events are located in the 3' untranslated region (3'UTR) between a stop codon and a polyadenylation site (PAS) of an mRNA. According to our current understanding, animal miRNAs follow this model and suppress protein-coding genes mostly by pairing with complementary \"target sites\" located in the 3'UTR, presumably leading to degradation of the transcript by cleavage or deadenylation , or the Target predictions have initially relied on ad-hoc UTR annotations , and aret al. recently introduced the additional category of \"composite\" terminal exons, i.e. exons which can either be internal and spliced to a more downstream one, or terminal due to suppression of the 5' splice site and polyadenylation in the downstream intron [We distinguish here two non-disjoint classes of alternative 3'UTRs Figure : Those am intron . AccordiWe based our annotation of 3'UTRs on the databases PolyA_DB  and RefSet al. [In this study, we restricted ourselves to the most established class of miRNA targets, those with complementary seed matches. We considered one particular prediction scenario with an estimated signal-to-noise-ratio of 3.8, which was one of several alternatives examined in detail by Lewis et al. : we requWe predicted 1,981 genes 27.7%) as being targeted by one or more miRNA, with an average of 3.6 target sites per targeted gene, and an average of 35.5 mRNA targets per miRNA seed. Figure 7.7% as bconstitutive targets, which encompass predicted target genes without alternative PAS, as well as genes with alternative PAS, but with all sites located within the constitutive UTR regions ; (b) on/off targets, which are genes having alternative PAS, and in which target sites fall exclusively into alternative UTR regions (469 genes = 23.7%); and (c) modulated targets, which contain genes with alternative PAS, and sites fall into both constitutive and alternative UTR regions (392 genes = 19.6%). Groups (b) and (c) together form the set of alternatively targeted genes in which one or more target sites are located in alternative 3'UTR segments. Altogether, a total of 43.3% of genes predicted as miRNA targets have at least some target sites which are likely to not always be part of the transcript.Targeted genes with alternative PAS may have target sites in constitutive and/or alternative UTR regions. In our set, 40% of the sites fell in alternative segments. To provide a more detailed picture of miRNA targeting, we classified target genes into three groups: (a) On/off targets are targeted by fewer miRNAs than other categories: They have an average target site density of 1.4 per kb , compared to 2.9/kb  for modulated genes. This is not an artifact of shorter on/off UTRs; the maximal UTR length distributions for both on/off and modulated targets are similar to the overall distribution of alternatively targeted genes , which is targeted by the muscle-specific miR-1 but has a muscle-specific alternative 3'UTR which excludes the miR-1 target sites [The expression of genes is fine-tuned and coordinated on a number of levels. The results in this study point to one piece in this puzzle of interactions among post-transcriptional regulatory mechanisms. In particular, it refines the notion of targets and anti-targets  examinedet sites . Inevitaafter internal alternative cleavage sites, which may be due to interactions of RNA processing mechanisms. We do know that this pattern is not caused by specific microRNAs or mRNAs and rather an overall more global phenomenon.It is at this point not clear what the causes for target site enrichment around polyadenylation sites are. It is conceivable that the interior of long UTRs is generally prone to form secondary structure, and thus be not as accessible to the RISC complex, than at the beginning and end. The depletion of target sites within the first 20 nucleotides immediately downstream of the stop codon suggests however that the very beginning of the 3'UTR region is a suboptimal area for targeting, as it may be obstructed by the ribosome complex. These observations do however not explain the enrichment The target prediction approach we used is strongly based on conservation, and we cannot completely exclude that our observations may be caused by artifacts, e.g. in case genomic alignments should perform better on the region around polyA sites than the rest of the UTR. Indeed, conservation plots of 3'UTRs , whether only Watson-Crick base pairs or also G-U base pairs are allowed, and whether the identification of conserved targets relies on pre-computed alignments or is carried out independently in each species. Accordingly, the number of predicted targets and the associated SNR will change somewhat. miRNAs are commonly grouped into families, the members of which share the same seed and are thus predicted to target the same genes by algorithms relying on seed complementarity only. We considered one particular prediction scenario of the TargetScanS algorithm with an estimated signal-to-noise-ratio of 3.8 : we requet al. described 5-way conserved predictions in 1,509 genes, we in 1,593, with an overlap of 852 genes (56%). It is easy to conceive that we made additional predictions due to the increased volume of available cDNA and EST sequences. In addition, of the 657 genes predicted by TargetScanS alone but not present in our analysis, 531 (81%) are not in our database of reliable complete 3'UTR annotations and are thus not predicted by default. Therefore, the predictions are in good agreement, but the discrepancies hint at the strong influence that reliable and complete UTR definitions have on the predictions of miRNA targets.At the time of the initial TargetScanS algorithm, only 62 miRNA families were known; restricting our predictions to the same set of families, we can assess how our analysis compares to this smaller earlier set. (More recent publicly available sets of TargetScan predictions are not based on the same group of five conserved species and thus not amenable for comparison.) Lewis A complete list of predictions is provided in Additional Files WHM implemented the UTR and microRNA target database. Both WHM and UO analyzed the data. UO conceived the study and wrote the manuscript.Tables with enriched Gene Ontology categories of miRNA targeted genes.Click here for fileConservation plots of mammalian 3'UTRs.Click here for fileList of targeted genes and coordinates of target sites, sorted by targeted gene.Click here for fileList of targeted genes and coordinates of target sites, sorted by miRNA.Click here for file"}
+{"text": "Biomphalaria glabrata is an intermediate snail host for Schistosoma mansoni, a medically important schistosome. In order to identify transcripts involved in snail-schistosome interactions, subtractive cDNA libraries were prepared, using suppression subtractive hybridization (SSH) between a parasite-exposed schistosome-resistant and a susceptible strain of B. glabrata, and also between schistosome-exposed and unexposed snails from the resistant snail line. Separate libraries were made from both haemocytes and the haemopoietic organ. Subtraction was performed in both directions enriching for cDNAs differentially expressed between parasite-exposed resistant and susceptible samples and up or down-regulated in the resistant line after challenge. The resulting eight libraries were screened and eight genes, differentially expressed between the haemocytes of resistant and susceptible snail strains, were identified and confirmed with reverse transcriptase PCR, including two transcripts expected to be involved in the stress response mechanism for regulating the damaging oxidative burst pathways involved in cytotoxic killing of the parasite: the iron-storage and immunoregulatory molecule, ferritin, and HtrA2, a serine protease involved in the cellular stress response. Transcripts with elevated levels in the resistant strain, had the same expression patterns in the subtracted libraries and unsubtracted controls; higher levels in exposed resistant snails compared to susceptible ones and down-regulated in exposed compared with unexposed resistant snails. Differential expression of two of the transcripts with no known function from the susceptible strain, was independently confirmed in a repeat exposure experiment. It is also an important model invertebrate and currently the subject of a large-scale genome project . Interactions between snails and schistosomes are complex and there is a need to elucidate pathways involved in snail-parasite interactions and identify those factors involved in the intricate balance between the snail internal defence system (IDS) and trematode infectivity mechanisms that determine the success or failure of an infection  and 60 from resistant strain  and stored at 4\u00a0\u00b0C until dissection. The haemopoietic organ, responsible for producing haemocytes that have an active role in the snail internal defence system  according to the manufacturer's protocol. This kit includes DNAse treatment to eliminate genomic DNA contamination. cDNA synthesis and subsequent amplification was performed using 1\u00a0\u03bcg total RNA with the SMART\u2122 PCR cDNA synthesis kit (CLONTECH) according to the manufacturer's instructions.2.3Eight independently subtracted libraries were made using the PCR select kit (BD Clontech). These were resistant minus susceptible and vice versa for both the haemopoietic organ and haemocytes from the first experiment and exposed minus unexposed snails of the resistant line and vice versa from the second experiment again both from the haemocytes and haemopoietic organ. The subtraction was carried out as described in the kit manufacturer's instructions. However, six primary amplification PCRs, followed by a secondary PCR from each primary PCR were carried out and then these PCRs were pooled for library construction. This multiple sampling of each subtracted pool was designed to minimize sampling effects, whereby transcripts may be present or absent by chance. The libraries were made by ligating 3\u00a0\u03bcl of each pool of secondary PCRs into pGem-T easy vector (Promega) and transforming JM109 cells according to the manufacturer's instructions. The entire library was plated out onto 10 LB agar plates. From each library 2\u00d7 96 white colonies were selected for overnight culture.2.4Duplicate filters were made for each library by spotting 2\u00d7 96 clones grown overnight in LB, onto Biodyne B membrane (PALL) using dot-blotting apparatus . The membrane was soaked in 2\u00d7 SSC prior to loading in the press. Then 50\u00a0\u03bcl 2\u00d7 SSC with dilute bromophenol blue was sucked through followed by 20\u00a0\u03bcl LB culture. The bacteria were then denatured with 50\u00a0\u03bcl denaturing solution , then neutralized with 50\u00a0\u03bcl of 1.5\u00a0M NaCl, 0.5\u00a0M Tris pH 7.5 solution for 2\u00a0min, and rinsed in 50\u00a0\u03bcl 2\u00d7 SSC. The filters were air dried and stored at RT.SmaI and RsaI (Boerhinger Mannheim) to remove adapter sequences and labelled with digoxigenin using DIG High Prime DNA labeling detection starter kit II (Roche). The filters were visualized by autoradiography. Positive spots were identified as those that were clearly present on the filter probed with the corresponding library while completely absent from the reverse subtracted library.For each pair of duplicate filters, one was probed with the library from which it was made, and one with the reciprocal subtracted library, so for example the haemocyte resistant specific library filters were probed with the haemocyte resistant specific library as a probe, as well as using the susceptible haemocyte library as a probe. In each case the probe used was 10\u201315\u00a0\u03bcl of cleaned secondary PCR, previously digested with the restriction enzymes, 2.5http://www.seqtools.dk/.) All sequences were submitted to GenBank (Accession numbers DY523246\u2013DY523271).All identified fragments were sequenced using BigDye and run on an ABI 377. The sequence traces were examined using Sequencher and all vector and adaptor sequences removed. Sequences were compared to GenBank using BlastN BlastX and tBlastX using the program Seqtools  was used to assess the comparability of samples for the unsubtracted control material to confirm that equivalent quantities of cDNA had been used for the specific primers. PCR and cycling conditions were the same as for specific PCRs. Since the subtracted libraries had non-differentially expressed genes (such as actin) subtracted from them, it is not possible to use this type of control for these samples.For each identified candidate specific primers were designed and synthesized. Semi-quantitative PCR used 0.4\u00a0\u03bcM Forward and Reverse primers see , 1\u00d7 advaFor the repeat experiment, 1\u00a0\u03bcl cDNA (prior to SMART amplification) was used and RT-PCR performed as above. For four of the transcripts, no amplification products were obtained from this cDNA so SMART-amplified cDNA was used as above. Actin PCRs were carried out simultaneously on both unamplified and SMART-amplified cDNA as a control. To assess statistically differential expression of these transcripts, integrated optical density (IOD) measurements were obtained for each amplified band, as a proxy for gene expression , and a matched paired t-test performed for each transcript, using log IOD values normalized to actin.33.1SSH was used to make eight subtracted libraries; forward and reverse subtraction of resistant and susceptible material from haemocytes and the haemopoietic organ made two resistant-specific and two susceptible-specific libraries, one for each tissue type; and from the second experiment, forward and reverse subtraction of parasite-exposed and unexposed material from the resistant line, again from the same tissue types, made two exposed-specific and two unexposed-specific libraries. Differential screening of the 8 subtracted libraries identified 24 differentially expressed positive candidate transcripts, all of which were sequenced. These candidates were selected by presence or absence on the filters , quantitNine of the candidate sequences originated from haemocyte material, eight of which were derived from the first experiment comparing exposed resistant and susceptible snail strains. Five different sequences from the resistant minus the susceptible library were identified; Clusters 2\u20135 and one single sequence ZB9413. Three positives were identified from the susceptible minus resistant library, two unique sequences (ZB9365 and ZBA105) and one cluster of 6 sequences (Cluster 1), which are transcripts specific to the haemocyte susceptible library. A single transcript (ZBA3283) was identified from the second experiment of resistant-exposed minus resistant-unexposed library.Only 2 clear positive transcripts were obtained from the haemopoietic organ material, one (ZBA2946) from the resistant unexposed minus exposed library, and one (ZB9039) from the resistant exposed minus susceptible exposed library.3.2E value of <1\u00a0\u00d7\u00a010\u22124 for a significant match, only four had significant BLAST hits in the non-redundant section of GenBank. Cluster 5 (six sequences) identified soma ferritin from Lymnaea stagnalisB. glabrata (AY028461) B. glabrata ESTs, and since this transcript also contained a poly(A) tail (more than 26\u00a0bp long), a poly adenylation signal (24\u00a0bp upstream from the start of the polyA sequence) and a 147 amino acid long open reading frame, the sequence is definitely derived from mRNA. The other three sequences from the resistant specific library identified no significant matches in the non-redundant section of GenBank, but all identified previously sequenced B. glabrata transcripts from dbEST. Cluster 2 initially identified 34 B. glabrata ESTs, but the resulting contig identified a further 11 ESTs producing a final 1493\u00a0bp contig composed of 45 ESTs, all Open Reading frame ESTs (ORESTES) CB350578) identified from a differential display experiment comparing the effect of S. mansoni and Echinostoma caproni excretory-secretory products on Bge cells, but not investigated further in that study B. glabrata ESTs and the slightly extended sequence had no additional significant hits. Of the three sequences from the susceptible library, only one (ZBA105) identified a sequence on the database, but this was a putative hypothetical protein from C. elegans, which provides no information as to the function of this protein. The sequence from the exposed-specific library from the resistant snail line, ZBA3283, did not produce a significant hit, but examining both the BlastN and BlastX results showed that all the sequences identified were mitogen activated protein kinase (MAPK), based on the first 120 bases of the transcript fragment. This translated section identified the 3\u2032 section of the catalytic domain of serine/threonine kinase with a BlastP search at NCBI entrez Blast interface (http://www.ncbi.nlm.nih.gov/entrez)  and suggests that the transcript fragment obtained from the SSH (which has a polyA tail) contains only the very end of the coding region of MAPK followed by the 3\u2032 UTR, and this coding section was insufficient to produce a significant Blast result. It seems probable that this is a MAPK-like sequence but more sequence would be required to confirm its homology to MAPK. One of the transcript fragments (ZB9039) from the haemopoietic organ showed no significant database match, while the other (ZBA2946) was significantly similar to a serine protease HTRA2, from many organisms including mouse and human All the sequences were compared to the non-redundant and expressed sequence tag (EST) sections of GenBank using BlastN, BlastX and tBlastX , again using semi-quantitative PCR on the subtracted and unsubtracted fractions obtained from the SSH.3.4.1The expression of the four transcripts with elevated expression in haemocytes from resistant exposed snails (compared with susceptible exposed strains) was independently examined in the exposed resistant compared with unexposed resistant subtractions . This sh3.4.2Of the fragments with elevated transcript levels in the susceptible strain identified from haemocytes  both Clu3.4.3The expression of ZBA2946 (serine protease HtrA2), the fragment identified from the unexposed resistant library from haemopoietic organ was also examined in the independently subtracted haemocyte material from the same experiment and this expression pattern seems to mirror that found in the haemopoietic organ material b. Howeve3.5Since all of the fragments identified demonstrated differential expression between resistant and susceptible snail haemocytes, this experiment was repeated and the expression of each transcript examined in the haemocytes of these snails . Using i4S. mansoni-exposed resistant and susceptible B. glabrata lines were created to identify defence-relevant genes that are involved specifically in the resistant response. We also examined parasite-exposed and unexposed resistant snails to see which genes were differentially regulated in response to S. mansoni infection. These aims were also addressed using haemopoietic organ (tissue responsible for producing the haemocytes) SSH libraries using the same snail lines and exposures. Differential screening of 8 subtracted libraries produced 11 sequence fragments that were strong candidates for differential expression. RT-PCR using the original subtracted and control material confirmed differential expression of eight of these transcripts, seven of which were derived from haemocyte libraries. Only four had significant Blast hits in GenBank, three from haemocyte libraries  and one from a haemopoietic organ library (serine protease HtrA2), while three show no homology to characterized genes on GenBank, although several do identify previously sequenced ESTs with no function ascribed to them. All the fragments demonstrating elevated levels in the resistant strain, had the same expression patterns in the subtracted libraries and unsubtracted controls; higher levels in exposed resistant snails compared to susceptible ones and down-regulated in exposed compared with unexposed resistant snails. Clear differential expression of two susceptible-specific fragments (Cluster 1 and ZB9365) with no known function was shown to be repeatable.In this study, haemocyte suppressive subtractive libraries of 4.1AY028461) B. glabrata ESTs, four of which contained this section of sequence that identified the FREP gene. No ESTs from other organisms were identified with this sequence. In some cases, however, these other ESTs did not align outside this region. Therefore another explanation for finding this sequence in the intron could be that it is a common B. glabrata genomic sequence motif. However, without recovering more sequence from this transcript and resolving or refuting its homology with FREP genes it is not possible to speculate further upon its function.Cluster 4 identified a section of sequence within intron 3 of one FREP molecule, a fibrinogen-related protein 3-2 precursor  of vertebrate ferritin mRNAs Cluster 5 had significant sequence homology to soma ferritin from the freshwater snail B. glabrata ESTs with homology to ferritin have been previously reported. Nowak et al. CD760630) from a head/foot SSH library from resistant B. glabrata 12\u00a0h post infection with S. mansoni which demonstrated little differential expression, and Raghavan et al. AW739853) from a resistant strain (BS-90) haemocyte library 5\u00a0h post-exposure to S. mansoni. These two sequences are identical for the 303\u00a0bp, which they both contain. However, Blast analysis with Cluster 5 revealed that it was different to these ferritin-like transcripts from earlier studies; although all identified the soma ferritin from L. stagnalis as the closest protein match, Cluster 5 sequence had only 80% identity with CD760630 (127/158\u00a0bp) and 79% with AW739853 (127/224\u00a0bp) from the Blast results. Cluster 5 did identify two other B. glabrata ESTs AW740322 and AW740403 from the haemocytes of unexposed BS-90 (resistant) snails B. glabrata ferritin gene. Two ferritin genes have also been identified in the oyster Crassostrea gigasL. stagnalis, Two Litterina littoreaHelix pomatia2O2 induces ferritin in HeLa cells B. glabrata the production of H2O2 demonstrated when the respiratory burst is artificially stimulated, appears greater in a resistant snail line 2O2, has been shown to have a constitutive difference in mRNA levels between one mostly resistant snail line and susceptible snails The function of ferritin in invertebrates is less well characterized than in mammals, but it has been shown to be upregulated by LPS in echinoderms 4.2.2Clone ZBA2946 sequence showed considerable homology to a mouse serine protease HtrA2 B. glabrata is unknown, but, given the function of homologous proteins in other organisms, it may be involved in the cellular stress response. Previous findings of serine protease activity in B. glabrata include Bahgat et al. B. glabrata haemocytes, a finding consistent with these phagocytic cells mounting a cytotoxic attack against schistosome sporocysts. However, although the serine protease's activity varied among individual snails, there was no detectable difference between snails susceptible or resistant to schistosome infection. One EST cluster (GenBank Accession no. CK989867) identified in a haemocyte cDNA library from snails maintained in non-axenic conditions The role of HtrA2 in 4.3Both ferritin and HtrA2, as stress response genes, would be expected to be stimulated in response to schistosome challenge, and elevated levels in resistant snails are consistent with this. How then to account for the reduction in mRNA for both these genes that appears in parasite-exposed resistant snails compared to unexposed snails? One possible explanation may lie with the interaction between snail and parasite. The influence of the parasite must be considered since it manipulates the normal snail defence response, and this effect may be greater in susceptible snails than in resistant ones. It is possible that some effect of the parasite that reduces the snails\u2019 normal response to oxidative stress occurs, but is less significant in resistant snails, allowing them to overcome the challenge. This would result in the expression patterns seen for these genes and would also account for the low levels detected in the repeated experiment.Two identified susceptible-specific transcripts (Cluster1 and ZB9365) were independently confirmed as being present only in susceptible snails in the repeat experiment. Further characterization of these transcripts with unknown function is required to determine if these transcripts are involved in the response of susceptible snails to parasite exposure and how that response differs to that of resistant snails. Surprisingly six of the identified candidates did not show differential expression in the repeat experiment. This could be due to a number of factors, including variability in the conditions of parasite exposure, loss of signal due to pooling the samples over the 24\u00a0h time period  and the fact that for four of the samples a product could only be detected faintly after amplifying the cDNA, which again might prevent the detection of small differences in expression.B. glabrata that are expected to be involved in the stress response mechanism for regulating the damaging oxidative burst pathways involved in cytotoxic killing of the parasite. While no single transcript is likely to be solely responsible for conferring differing susceptibility to S. mansoni infection, differences in gene expression demonstrate a difference in the snails\u2019 response to infection and may indicate which mechanisms are employed by the snails in their defence response. Whilst interesting candidate genes have been found using SSH, it is not a particularly efficient method for the analysis of gene expression, especially given that many of the candidates identified in this way did not show differential expression in a repeat experiment. A different approach to transcriptome analysis, such as using microarray technology, will enable a more thorough examination of changes in gene expression in B. glabrata, as a much larger number of genes can be analyzed simultaneously, using smaller amounts of RNA. The SSH libraries produced in this study, as well as larger libraries from a modification of the EST strategy, termed ORESTES (Open Reading Frame ESTs; B. glabrata cDNA microarray. This will enable a more detailed investigation of the transcriptome in response to trematode infection in this snail intermediate host. This approach has been used successfully for expression studies of gender-associated gene transcripts in S. mansoniIn summary, we have identified several transcripts by differential screening of SSH libraries from"}
+{"text": "Until recently, few genomic reagents specific for non-human primate research have been available. To address this need, we have constructed a macaque-specific high-density oligonucleotide microarray by using highly fragmented low-pass sequence contigs from the rhesus genome project together with the detailed sequence and exon structure of the human genome. Using this method, we designed oligonucleotide probes to over 17,000 distinct rhesus/human gene orthologs and increased by four-fold the number of available genes relative to our first-generation expressed sequence tag (EST)-derived array.We constructed a database containing 248,000 exon sequences from 23,000 human RefSeq genes and compared each human exon with its best matching sequence in the January 2005 version of the rhesus genome project list of 486,000 DNA contigs. Best matching rhesus exon sequences for each of the 23,000 human genes were then concatenated in the proper order and orientation to produce a rhesus \"virtual transcriptome.\" Microarray probes were designed, one per gene, to the region closest to the 3' untranslated region (UTR) of each rhesus virtual transcript. Each probe was compared to a composite rhesus/human transcript database to test for cross-hybridization potential yielding a final probe set representing 18,296 rhesus/human gene orthologs, including transcript variants, and over 17,000 distinct genes. We hybridized mRNA from rhesus brain and spleen to both the EST- and genome-derived microarrays. Besides four-fold greater gene coverage, the genome-derived array also showed greater mean signal intensities for genes present on both arrays. Genome-derived probes showed 99.4% identity when compared to 4,767 rhesus GenBank sequence tag site (STS) sequences indicating that early stage low-pass versions of complex genomes are of sufficient quality to yield valuable functional genomic information when combined with finished genome information from a closely related species.The number of different genes represented on microarrays for unfinished genomes can be greatly increased by matching known gene transcript annotations from a closely related species with sequence data from the unfinished genome. Signal intensity on both EST- and genome-derived arrays was highly correlated with probe distance from the 3' UTR, information often missing from ESTs yet present in early-stage genome projects. Macaca mulatta) serves as a model for many facets of human development and physiology and is one of the most widely used nonhuman primates for the study of infectious diseases, such as AIDS. The widespread use of this species in biomedical research led to a proposal in 2002 to generate its complete genome sequence [The rhesus macaque  . Over 36For the above reasons, there was clearly a need for an alternative to our EST-based array to provide better coverage of rhesus/human gene orthologs and to optimize probe selection regions to include genomic regions proximal to the 3' end of gene transcripts. In the present study, we employed a human/rhesus comparative genomics approach to address these issues, using human genome sequence and annotation to derive optimal probe design regions from an unfinished and highly fragmented build of the rhesus genome sequence. This approach greatly increased the number of genes available for expression analysis and optimized hybridization signal intensities. These results show that early stage genome projects are a valuable source of information that can be immediately utilized for functional genomics assays.Earlier studies report that the terminal exon of human transcripts, which contains the optimal 3' UTR microarray probe design region, averages almost 1,400 bp with a median length of 1,000 bp . If probWe matched human genome transcript information to rhesus unfinished contigs by initially comparing 22,975 terminal exon sequences derived from RefSeq with 486,000 Baylor rhesus genome project version 0.1 WGS contigs averaging 5,540 bp in length, excluding rhesus assemblies containing highly repetitive elements. Remarkably, we were able to match 22,797 out of the 22,975 human terminal exon sequences (99%) to the unfinished rhesus genome sequence, including 17,702 out of 17,934 (99%) distinct rhesus/human RefSeq gene orthologs. Mean alignment size was approximately 1,100 bp covering greater than 90% of each human terminal exon. Human terminal exon sequences matched rhesus contigs with a 94.4% mean identity.Microarray design requires comparing all individual probes with all transcriptome sequences to exclude probes highly similar to more than one location in the transcriptome and therefore subject to non-specific mRNA cross-hybridization . To addrWe tested the similarity of the KRM2 genome-derived probes to other available rhesus sequences to gauge the quality of WGS contigs used in probe design. We searched each KRM2 probe against a BLAST database of 4,767 high-quality GenBank STS sequences derived from rhesus PCR products designed specifically to 3' UTR regions . We founFigure n = 4). The same probes were also used to replicate the experiment on our EST-based KRM1 microarray. Brain and spleen tissues were chosen for the experiment to maximize potential differential expression of KRM1 probes, which were mainly designed using rhesus brain and spleen tissue EST sequences. A minimum two-fold difference in test/reference channel signal intensity and a maximum P value of 0.01 were selected as cut offs to indicate differential gene expression for individual probes.We tested the KRM2 microarray by hybridizing cRNA probes generated from equal mass amounts of mRNA derived from rhesus brain and spleen  showed differential expression on the KRM2 array. The greater number of differentially expressed genes on KRM2 versus KRM1 for genes represented on both arrays suggests a distinct improvement in probe performance using genomic versus EST based array probe design. Despite this improvement, overlap in expression results between identical genes on KRM2 and KRM1 is, as expected, not perfect, and microarray results will continue to be used, by our lab and others, as part of a discovery process requiring individual gene verification by methods such as RT-PCR. Hybridization results for all rhesus probes for both arrays in the above experiment are given in supplemental Tables 1 and 2 [see Additional files The results of the brain versus spleen hybridizations highlight the extra coverage garnered in the KRM2 array. Figure One of the greatest benefits of utilizing early stage genome information in conjunction with closely related species annotation in oligonucleotide microarray design is the huge increase in the number of different genes available for mRNA abundance measurements compared with relying on EST sequence data alone. This increased number of genes in turn helps provide more comprehensive input for pathway and network analysis of differential gene expression. Figure The relationship between signal intensity and probe distance from the 3' UTR was much more dramatic than expected and a factor to be seriously considered in microarray probe design as well as microarray analysis. The apparent reason for this bias appears to relate to the efficiency of the reverse transcriptase reaction.M. nemestrina and M. fascicularis, can also be used for gene expression analysis on the rhesus genome-based KRM2 microarray. 18,296 KRM2 rhesus probes compared with M. nemestrina and M. fascicularis EST sequences showed a greater than 98% similarity to both species, representing on average a single base mismatch per 60-mer probe.Historically, full-length cDNA transcripts have been challenging to achieve, with most first-strand synthesis reactions resulting in pools of transcripts in the 400 bp to 1.5 Kbp range. Premature terminations of the reaction frequently occur due to secondary and tertiary structures in the transcript, and increased product size is often associated with increases in single-base errors in the sequence . Due to Although there are many advantages in leveraging annotations from closely related species to design microarray probes from genomic sequences, there are obvious limitations to this method. Gene isoforms, including splice variants, are restricted to those mapped from the related species annotation; true splice variant information still has to be obtained by other methods, such as cDNA sequencing from different tissues from the microarray target species. Species-specific genes are also not addressed using this method. Important innate immunity genes, such as theta-defensins, are only expressed in Old World monkeys such as rhesus macaque , whereasIn addition to designing rhesus probes using human genome annotations, we will also continue to use species-specific cDNA-derived probes on future macaque microarrays, particularly in light of the growth of the number of macaque ESTs and full-length mRNA transcripts available in public databases. Currently, almost 1,000 EST-derived probes were carried over from KRM1 onto the new KRM2 microarray. Ultimately, microarray probes derived from macaque-specific unannotated assemblies may be the most interesting of all, since they offer the greatest potential for discovering new genes and gene expression pathways .Figure -4. Human/rhesus High Scoring Pairs (HSPs) for the best matching rhesus contig for each exon were parsed from BLAST output and stored in an exon alignment table. This table was used as input to a Java script to determine the mapping between BLASTN human exon HSPs and the best matching rhesus genome contig for each human exon. The longest matching rhesus HSP was used to establish the initial probe target alignment region between each human exon and the best matching rhesus contig. Additional rhesus HSPs were used to extend the alignment region upstream and downstream from the initial alignment until all HSPs were used. Once a rhesus contig alignment region was established, the DNA sequence for this region was used to produce a full-length alignment with its corresponding human exon DNA sequence using CLUSTALW. Ungapped full-length rhesus genome DNA alignment regions for each gene were then handed off to the probe design process as probe design target sequence candidates. Terminal exons were used whenever possible for probe design due to their proximity to the 3' end of a gene and because they do not cross intron/exon DNA sequence boundaries. A gzipped fasta file of rhesus DNA alignment regions matching terminal exons for 22,797 human RefSeq genes produced by the bioinformatics pipeline using the above process is provided in A MySQL database table was constructed to store 248,000 NCBI RefSeq exon sequences and exon positions for 23,000 human RefSeq genes. Each human exon sequence was searched using BLASTN against a database of 486,600 Baylor rhesus genome contigs with BLAST parameters of b = 1, v = 1 and expected value of 10in situ oligonucleotide microarray platform. Final probes were selected from the candidate set based upon duplex formation stability and cross-hybridization potential with a defined transcriptome database.Probes were designed using the commercial probe design process and associated algorithms of Agilent Technologies, Inc. As part of this process, all replicate target sequences were removed along with sequences that were too short for probe design. Target sequences were then vector masked using an Agilent-modified version of the Univec database  in conjuA probe design file was created to distribute the probes randomly on an Agilent 22K-featured microarray. The final array design (AMADID# 013791) is available for review and download free of charge for registered users of the Agilent eArray application . A complSpleen and brain tissues were collected at necropsy from healthy adult rhesus macaques and immersed immediately in RNAlater (Ambion Inc.) to preserve the quality of RNA.Brain tissue was pooled from cerebreum and cerebellum from two macaque females and spleen tissue was pooled from four macaque females and one macaque male.Tissue samples were then homogenized with Solution D . Homogenization with a Kinematica Polytron PT1200 instrument and the model PT-DA1212/2 generator  lasted for 30 seconds in 10 ml round-bottom polypropylene test tubes with 5 ml of solution D. In order to reduce generation of aerosols during this process, the polytron's generator was passed through a hole in the test tube lid that had been drilled in a manner that ensured a tight fit with the instrument. To further minimize possible contact with aerosols, a barrier shield was used in addition to positive air pressure respirators and full protective personal protection equipment. Total RNA was subsequently extracted using RNeasy columns (Qiagen) and the quality and quantity of the total RNA was determined by using a Nanodrop spectrophotometer (Nanodrop Technologies) and the Bionalyzer 2100 system (Agilent Technologies).P \u2264 0.01) and an expression level change of 2 fold or greater. Finally, biological gene sets (referred to as Biosets) were compiled for key cellular processes by selecting genes of interest that were both represented on the microarray and which had Gene Ontology (GO) annotation. In accordance with proposed standards [Samples of both the spleen and brain tissues were pooled using equal amounts of total RNA. cRNA target production was done with the Agilent Low RNA Input Fluorescent Linear Amplification kit (Agilent Technologies). Slides were scanned with an Agilent DNA microarray scanner, and image analysis was performed using Agilent Feature Extractor Software. Each microarray experiment was done with two technical replicates by reversing dye hybridization for experimental and reference samples. All data were entered into a custom-designed database, Expression Array Manager , and thetandards , all dattandards . Microartandards  accessioJCW was responsible for all algorithm, software and database development and computational analyses used in the microarray bioinformatics pipeline up to the final probe-design process. JCW, MJK, and BP were responsible for drafting and editing the manuscript. SCP and MJT provided biological materials for array hybridization and subsequent data analysis. CLM and SPI provided macaque EST sequence data and associated analyses. CN led the final probe-design process for the KRM2 array. MGK conceived of and coordinated the study and gave final approval of the version to be published. All authors read and approved the final manuscript.KRM1 hybridization analysis. A Microsoft Excel table showing fold-change and pvalues for brain versus spleen tissues for the KRM1 microarray hybridization results described in this study.Click here for fileKRM2 hybridization analysis. A Microsoft Excel table showing fold-change and pvalues for brain versus spleen tissues for the KRM2 microarray hybridization results described in this study.Click here for fileHuman/rhesus 3' UTR alignments. A FASTA format file of rhesus DNA sequence alignment regions matching terminal exons for 22,797 human RefSeq genes used as KRM2 probe design candidate regions.Click here for fileKRM2 probes. A Microsoft Excel table of KRM2 gene names, 60-mer rhesus DNA probe sequences and base pair distances from the 3' UTR of corresponding human RefSeq gene transcripts.Click here for file"}
+{"text": "Here we describe PathogenMIPer, a software program for designing molecular inversion probe (MIP) oligonucleotides for use in pathogen identification and detection. The software designs unique and specific oligonucleotide probes targeting microbial or other genomes. The tool tailors all probe sequence components  and combines these components into ready-to-order probes for use in a MIP assay. The system can harness the genetic variability available in an entire genome in designing specific probes for the detection of multiple co-infections in a single tube using a MIP assay.Akhras et al, personal communication).PathogenMIPer can accept sequence data in FASTA file format, and other parameter inputs from the user through a graphical user interface. It can design MIPs not only for pathogens, but for any genome for use in parallel genomic analyses. The software was validated experimentally by applying it to the detection of human papilloma virus (HPV) as a model system, which is associated with various human malignancies including cervical and skin cancers. Initial tests of laboratory samples using the MIPs developed by the PathogenMIPer to recognize 24 different types of HPVs gave very promising results, detecting even a small viral load of single as well as multiple infections (PathogenMIPer is a software for designing molecular inversion probes for detection of multiple target DNAs in a sample using MIP assays. It enables broader use of MIP technology in the detection through genotyping of pathogens that are complex, difficult-to-amplify, or present in multiple subtypes in a sample. Detection of the different types and subtypes of a pathogen is very important in clinical diagnosis. Over the past several years, the development and application of molecular diagnostic techniques has initiated a revolution in the diagnosis and monitoring of infectious diseases. Advances in molecular and cell biology have provided us with an understanding of the mechanisms of disease at the molecular level that can now be translated into designing new diagnostic, prognostic, and therapeutic tools. It is now possible to genotype/subtype pathogens to increase the accuracy and reproducibility of diagnosis. Detection of microbial DNA can provide not only a signature for the presence of a disease, but may also indicate a drug-resistant genotype, a particularly virulent subtype, a subtype associated with other clinically important sequelae, or the presence of multiple subtypes. These advances should open the way for administering more effective and efficient treatment for the control, prevention and cure of diseases. There are several multiplex detection methods currently in use, like DNA microarray, MUCH-AMASE, multiple sequencing and pyrosequencing -5, but tMolecular Inversion Probe (MIP) technology was initially developed for the detection of single nucleotide polymorphisms (SNPs) in human genes ,18. MIP A molecular inversion probe is comprised of genomic recognition sequences, common amplification sequences and a molecular barcode for each genotype assigned to a specific gene. This probe is a linear oligonucleotide with target-complementary sequences at the ends and a non-complementary linking segment in between Figure . Upon hyIn order to target the entire genome of a pathogen, the probes must be very sensitive, and long enough to detect even a few copies of the pathogen DNA in the sample. The reliability of the MIP assay depends wholly on the specificity and binding efficiency of the probes. To harness MIP technology and make it available for widespread clinical use, it will be necessary for researchers to rapidly design high quality probes. The classical approach for designing oligonucleotides, that is found in existing softwares -24 were A molecular inversion probe Figure  consistsThe software first generates target sequences and then in several consecutive steps eliminates unfit candidates. In order to minimize the time required to perform a design task, oligos are processed in several steps of increasing computational intensity, with a recursive elimination of unfit candidate probes at each of successive steps. The processing steps in our MIP design are as follows:1. Candidate probes for each of the target sequences are generated with a userspecified binding length.2. Candidates with a continuous stretch of six or more of the same base are eliminated.3. The middle base is checked for the preferred base (A|G|T|C). Candidates without the preferred middle base are eliminated.4. The remaining candidates are checked against non-target genomes. If an absolute match is found, the candidate is eliminated.5. The middle 11-base region is checked against other genomes to ensure uniqueness of probes and specificity of the assay.Automatically, the nonspecific candidates previously identified for other targets are also removed.6. Candidates are checked for their melting temperature (Tm) and those that are beyond the desired range are eliminated.7. Candidate probes whose similarity to non-target sequences exceeds a userspecified maximum are removed.8. The remaining probes are screened against the potential background caused by the host genome using an online BLAST search [9. In the final step, the tags are added to complete the design of ready-to-order probes. The tags are checked against the binding segments of all probes, to make sure that the addition of tags does not create a new recognizable sequence that may produce noise, false positives, or background during target detection.Initially (step 1), all sequences of the target with the specified binding length are considered as candidate probes. Each target-specific sequence is first examined for the presence of poly As, Cs, Gs or Ts and candidates with more than 6 repeats are eliminated (step 2). After completing the check of the middle base (step 3) and an initial search to eliminate homology with non-targets (step 4), the middle 11 base region of the probe, is checked against a non-target sequence (step 5) and those with a continuous stretch of similarity with the non-target sequences are eliminated (step 6). In step 7, the candidates are checked for their Tm using the nearest neighbor method -11. SurvEach target sequence will have a pool of anywhere from zero to a several thousands of probe candidates. The best of the unique candidates are selected from each pool by a selective filter tool step 8). In step 8, each of the candidates is scored for number of matching bases with the non-target sequences using a BLAST search [. In stepThe design strategy used in our software evaluates specificity at each step. Many existing oligo design tools -16 are dIn order to design a sensitive, reliable and consistent MIP assay, all probes for each target genome in the assay must be very selective and the selectivity determinants should be located mainly in the middle of the target binding region. These criteria are not enforced in any of the existing tools. The problem is made more complicated in a real biological situation, as some parts of the genome and of the target(s) may be highly mutated, so that even probes that are designed for a consensus target with high specificity may fail to bind to the actual targets, because of natural variation. Thus a pathogen MIP design strategy should include a user option to avoid particularly variable regions of the genome during the design of target-specific probes. The PathogenMIPer software allows for such options.PathogenMIPer designs candidate probes for each target sequence through several computational steps as is shown in Figure We used the PathogenMIPer software to design MIPs for 105 different types of human papilloma viruses (HPV). These genomes differ only slightly at most of the regions. Input constraints included a Tm of 60\u00b0C, length of 50 bp and middle base G. Multiple probes were designed for each target pathogen, and these probes complement different regions of the genome in order to exploit the variability of the whole genome and detect even small copies of the pathogen in the sample. In a preliminary test, twenty-four of the resulting probes  were added and the mixture was incubated for 60 minutes at 37\u00b0C and 20 minutes at 80\u00b0. Following exonucleolysis, the reaction was subjected to uracil depurination and cleavage with 2 units uracil-N-glycosylase  in 20 \u03bcL of 1.6 mmol/L MgCl2, 10 mmol/L Tris-HCl (pH 8.3), and 50 mmol/L KCl and incubated for 60 minutes at 37\u00b0C and 20 minutes at 80\u00b0C. To amplify the inverted probes, 1.5 units AmpliTaq Gold (Applied Biosystems), 10 pmol of each universal primer in 25 \u03bcL of 1.6 mmol/L MgCl2, 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, and 112 \u03bcmol/L deoxynucleotide triphosphate (dNTP) were added to the genotyping reactions.The samples were subjected to PCR using GP5/GP6+ primers, followed by pyrosequencing . The metThe reactions were amplified in 35 cycles of 95\u00b0C for 30 seconds, 55\u00b0C for 30 seconds, and 72\u00b0C for 10 seconds.Microarrays were prepared using CodeLink activated slides  with 5'amine-modified oligonucleotide. Oligonucleotides were printed onto microarray slides from a solution containing 1\u00d7 printing buffer , 20 \u03bcM probe using a microarrayer . Positive and negative controls were printed on the microarray as well.Each sample was printed in 4 replicates and 2 arrays were present on each chip. The post-printing processing of the microarray-chips were performed as recommended by the slide manufacturer.For the target hybridization step we used 50 \u03bcL of biotinylated PCR products, 1\u00d7 hybridization buffer  and 1.25\u00d7 Denhardt's solution. The hybridization was performed at 42\u00b0C for 12\u201316 h. After hybridization, the microarray was washed with wash buffer  2 times at 50\u00b0C for 2 min and once at room temperature for 2 min. The microarray was labeled for 10 min at 50\u00b0C with a solution containing streptavidin-allophycocyanin conjugated (1 mg/ml), 6\u00d7 SSPE, 1\u00d7 Denhardt's solution and 0.01% Tween 20. Following washing, 3 times in wash buffer, the microarray was assayed for fluorescent intensity at 635 nm using a GenePix 4000 fluorescent scanner  set to scan at 450 PMT. We used GenePix Pro software to determine the total fluorescent signal from each feature. To further confirm the results, 10 \u03bcl of the same PCR product that was used in microarray hybridization was sequenced by pyrosequencing as described by Gharizadeh et al [The sensitivity (Sn) and specificity (Sp) of any system can be defined in terms of four outcomes \u2013 true positive (TP), true negatives (TN), false positives (FP), false negatives (FN) ,18. In oAs described above, candidate homologous sequences were checked and evaluated, and the bad ones were eliminated (steps 1\u20136). Then, the average melting temperature of the candidates was calculated to check whether it was feasible to design MIPs with the specified Tm. Next, all candidates were checked for the middle base, and then for their Tm. The software designed total 4244 candidates for 105 HPV types, for a user specified Tm of 60\u00b0C (the software used 60 \u00b1 4\u00b0C), length 50 bp and middle base G. On an average 40 candidates were generated for each of the 105 HPV type genomes. The number of candidates generated for each genome varied from 30 to 75 depending on their genomic variability.As described above, in steps 4\u20136, the candidates for each HPV genotype target were checked against non-target genomes for their similarity. We checked the candidates of each HPV type against the genomes of other HPV types for similarity. The two homologous sites of the probes were evaluated in such a way that if they had any sequence matches in a non-target genome in the middle region, they incur much higher penalties than for the matches distal to the middle region. For each of the comparisons of the homologous sites of the probe against a non-target-genome, the total penalty score is checked against a threshold cut off value, set by the software based on the minimum mismatch percent specified by the user while setting up parameters for the design of MIPs. If the score exceeded the cutoff, the candidate was eliminated. When this filter was used against the candidate HPV probes generated in the previous step, PathogenMIPer returned a total of 2245 candidates, or 52% of the original candidates. The remaining 48% of the candidates were discarded in this step due to potential non-specific binding with non-target-genomes. The number of candidates returned per genome varied from 3 to 41 at the end of filtering.Finally (Steps 7\u20139), the best candidates were checked against the potential background or host genome through a remote BLAST and genome database on the NCBI server. Non-specific binding was reduced by making sure that the target-specific probe would not have more than 80% matches to the non-target genomes, or in their potential host genome. For the probes developed against 105 HPV types, the filtered candidates were subjected to BLAST against the human genome , and PathogenMIPer eliminated 61 candidate sequences based on the matches in host genome, rendering more specificity for the assay. The software returned 2184 candidate probes for all the 105 types of HPV.Once the homologous site construction is completed, the user can add predefined tag sequences to the homologous constructs. These tag sequences can be universal primers for the amplification of the binding signal, and/or molecular barcodes for the detection of the targets using tagArray  or by sequencing methods such as pyrosequencing .The tag sequences input by the user are added in the preferred order as shown in the figure The MIP assay was performed in 7 steps as depicted in figureSoftware performance of probe design is mainly limited by the amount of available memory, and the processor speed. The initial phase of generating candidate homologous sites (Steps 1\u20134) takes considerable processing time. In the example described here, this stage took ~3 hours. If more constraints are applied, the processing time will be increased, as more and more testing processes are done on the probe candidates. The number of targets and the length of each target affects the processing time as well. The total processing time is also depending on the number of candidates returned in the first step. When the number of targets increases, the processing time increases exponentially. Another major factor for the processing time is the divergence of the pathogen genomes. When the genomes of pathogens included in a MIP assay are more divergent, or distant, a greater number of candidates are generated and the processing time is longer.The second phase of evaluating the candidates (Steps 5\u20137), is a very computationally intensive process, and the time taken for this step (~50 minutes in our case) also depends on the parameters specified by user. The last step, in which the sequences are checked against the host genome through a BLAST, was done on the genome database at NCBI server. The criteria for BLAST are set by the PathogenMIPer software and the BLAST software called upon is the current version running remotely on the NCBI server.  Although this takes additional processing time (~9.5 hours in this example), it adds to the quality of the MIP assay by increasing uniqueness and reducing noise.When used for designing 105 different types of human papilloma viruses, PathogenMIPer generated a complete pool of probe sequences in less than 15 hours. The software is quite flexible in allowing candidates generated in each of the steps to be viewed, checked and accessed for editing so that one can go back to any step in the process and revise the design without deleting the candidates of the previous design, although such interventions would of course be more time-consuming.In preliminary tests with just 24 probes against 24 HPV genotypes, (i.e. one probe for each genotype present), we were able to sensitively detect all 24, with a lower limit of detection for purified plasmid as low as ~1 pg. In addition, we were able to detect multiple infections in samples containing HPV plasmids, without false positives, resulting in a specificity of 100%. Note that although PathogenMIPer designed 3 \u2013 20 probes for each HPV type, this initial validation used only one probe per genotype. By relying on a single probe for the entire genome of HPV, which is ~8000 bp long, poor binding or even non-binding of the probe to the target would result, if mutations occur in the same locus chosen for the design of the MIP for these genotypes, leading to suboptimal results. A more complete diagnostic would necessarily anticipate such a problem by incorporating multiple probes for those subtypes whose genetic variation makes their identity more difficult to distinguish.The PathogenMIPer system is extendable, with the addition of new modules and new design strategies. Because the quality requirements and other criteria for probes vary for different MIP assays, this software is able to support the design of different types of oligonucleotides, based on the constraints specified by the user. Although the processing time initially increases exponentially with the number of target genomes, this processing time levels off after a certain number of targets/genomes, as the software designs candidates by progressive elimination of non-specific candidates.in silico multiplex test system, whereby one can check the feasibility of designing a MIP assay for the specific targets one might want to include in a multiple detection technique. In the process of identifying unique specific sequences and adding tag sequences, universal primers, or cleavage sites to the complete probe design for a MIP assay, PathogenMIPer performs an efficient search for probes that have the highest probable success of producing a viable and robust MIP assay.Although the PathogenMIPer software is a tool for the design of highly specific oligonucleotides for use in MIP assays, this system essentially serves as an in silico system prior to launching costly and time-consuming laboratory experiments. The system can be extended to design probes for new types of assays such as hairpin inversion probes. Extending the software by adding various modules for designing different types of primers and probes is under way.PathogenMIPer is a software for designing molecular inversion probes for detection of multiple target DNAs in a sample. This software simplifies development of MIP assays, enabling broader use of MIP technology in the detection through genotyping of pathogens that are complex, difficult-to-amplify, or present in multiple subtypes. PathogenMIPer facilitates large scale multiplex assays, without the need for PCR sample preparation before analysis. For multiple detection strategies where amplification by a common primer is not possible, the feasibility of developing a MIP assay can be interrogated through our PathogenMIPer software is available as an additional file with the article.(See Additional files Name: PathogenMIPerOS: WindowsLanguage: PerlRequirements: Internet connection for background checking.ST designed and developed the software tool and drafted the manuscript. MK checked the software and helped to draft the manuscript. MA did the wet lab for validating the probes designed with PathogenMIPer software. RD helped to draft the manuscript. NP conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.This lists all the instructions for downloading and running the software.Click here for fileThis contains all the data files used for designing probes with the software and a subdirectory containing the result files obtained.Click here for fileIt has two software executables, needed for running the software.Click here for fileIt has two software executables, needed for running the software.Click here for fileIt has two files, needed for running the software.Click here for file"}
+{"text": "A genome-wide microarray analysis of sex-specific expression of alternative transcripts in Drosophila shows sexual dimorphism in transcript abundance for 53% of the genes. Drosophila melanogaster.Many genes produce multiple transcripts due to alternative splicing or utilization of alternative transcription initiation/termination sites. This 'transcriptome expansion' is thought to increase phenotypic complexity by allowing a single locus to produce several functionally distinct proteins. However, sex, genetic and developmental variation in the representation of alternative transcripts has never been examined systematically. Here, we describe a genome-wide analysis of sex-specific expression of alternative transcripts in Drosophila lines  using a newly designed 60-mer oligonucleotide microarray that allows us to distinguish a large proportion of alternative transcripts. The new microarray incorporates 7,207 oligonucleotides, satisfying stringent binding and specificity criteria that target both the common and the unique regions of 2,768 multi-transcript genes, as well as 12,912 oligonucleotides that target genes with a single known transcript. We estimate that up to 22% of genes that produce multiple transcripts show a sex-specific bias in the representation of alternative transcripts. Sexual dimorphism in overall transcript abundance was evident for 53% of genes. The X chromosome contains a significantly higher proportion of genes with female-biased transcription than the autosomes. However, genes on the X chromosome are no more likely to have a sexual bias in alternative transcript representation than autosomal genes.We compared transcript profiles in males and females from eight Drosophila suggests that a new level of sexual dimorphism at the molecular level exists.Widespread sex-specific expression of alternative transcripts in  Microarray hybridization, with its unprecedented ability to monitor genome-wide gene expression profiles, is paving the way for exploring previously intractable problems in developmental biology -5, neuroDrosophila whole-genome microarray that allows us to distinguish multiple transcripts of many genes using long (60-mer) oligonucleotide probes. Since genome annotation changes frequently as more data become available, we have created a flexible, easily updated design, and developed software that allows automatic annotation updates. We have used the new platform to compare gene expression profiles of males and females in eight lines of Drosophila melanogaster, and found that over 50% of all genes are expressed in a sex-biased manner. Interestingly, we estimate that between 11% and 24% of Drosophila genes known to produce multiple transcripts show sexual bias in the expression of alternative transcripts.To estimate the extent of sexual dimorphism and genetic variation in the production of alternative transcripts, we designed a new D. melanogaster, OregonR and 2b, and six randomly chosen recombinant inbred (RI) lines derived from these parents. We detected 8,292 genes with a single known transcript, represented by 8,310 microarray probes, in at least one line/sex combination. In addition, an additional 1,651 multi-transcript genes and 71 gene families were each represented by a single hybridizing probe, since some of the probes targeting alternative transcripts and gene families were not detected in this experiment. These 10,014 transcripts were analyzed using the ANOVA model for single transcripts . Of these transcripts, 56% showed significant variation at a false discovery rate (FDR) of 0.05 (Table CG33092) showed a significant difference in the interaction between line and sex.RNA was extracted from male and female flies from two laboratory lines of modulo), the direction of the difference between males and females was affected by genotype.For 828 of the 2,479 genes known to produce multiple transcripts, microarray probes targeting 2 or more distinct sets of transcripts showed detectable hybridization. These probes were analyzed using the ANOVA model for multiple transcripts. Expression levels of 653 (78%) of these genes showed significant variation at the FDR of 0.05, with the majority (544) showing a sex bias and 202 showing significant differences among lines . For 91 gene families, hybridization was detected for probes targeting two or more sets of transcripts. Of these, 79 were variable, with 67 of these showing significant differences between males and females. For one transcript , which are expressed in both sexes. tra2 was represented by four hybridizing probes that targeted different regions of a nearly identical set of transcripts; all of these probes showed similar ratios of expression in males and females (Table doublesex (dsx) is spliced in a sexually dimorphic manner, producing a male-specific and a female-specific transcript [dsx was represented by four probes: one targeting a male-specific exon, one targeting a female-specific exon, and two targeting an exon common to male and female transcripts. We found that the male-specific probe indeed showed male-biased expression, the female-specific probe showed female-biased expression, and the common probes showed expression levels intermediate between the two sex-specific probes  to examine the genes for which at least two probes targeting distinct sets of transcripts produced detectable hybridization. For these genes, we tested whether the relative amounts of signal from the different probes differed between sexes or lines. Such differences  imply that the same gene produces alternative transcripts in different amounts in males versus females, or in different genotypes, respectively.Next, we retrieved from FlyBase a list of genes known to be involved in the development or function of reproductive organs. We subdivided this list into three non-overlapping sets: genes known to function only in the female reproductive system , those known to function only in the male reproductive system (60 probes/42 genes), and genes implicated in both male and female reproductive systems (120 probes/86 genes). Most of these genes, however, are not exclusive to the reproductive system and are expressed in a wide range of non-reproductive organs as well. Since our experiments utilized whole-body RNA samples, we may not always be able to detect sex-biased expression in the reproductive organs. We found that among the female reproductive system genes, 86% were female-biased, with 72.5% being significant for sex and/or sex-by-probe interaction effect . Conversely, among the male reproductive system genes, 64.3% were male-biased, with 55.5% showing significant sex effect . We also analyzed a set of genes that are thought to be expressed only in males. These genes included a number of secreted accessory gland proteins -31, putaDrosophila is the dsx gene [dsx show different expression levels in different sexes , which also produces male- and female-specific alternative transcripts [Sex-specific production of alternative transcripts has previously been reported for only a handful of genes, so we lack an extensive set of positive controls against which to compare our results. The best-known example in dsx gene . Indeed,es Table . When annscripts , was repWe examined 828 genes for which 2 or more probes representing distinct sets of transcripts showed detectable hybridization. Of these, 182 (22%) showed significant sex-by-probe or line-by-probe interactions at the FDR of 0.05, indicating that the relative amounts of alternative transcripts were different in males and females, or in different lines Table . For theTo examine sex-specific expression of alternative transcripts more closely, we analyzed the set of 177 genes that showed significant sex-by-probe interactions on a probe-by-probe basis . In general, we found that probes targeting the same exon, or different constitutively spliced exons, tended to have similar male/female expression ratios . Such exceptions could be due either to intrinsic biases in probe hybridization, or to mistakes in the current FlyBase annotation . To estimate the extent to which our results may be affected by these factors, we used the ANOVA model for multiple transcripts to compare probes that, according to the current annotation, targeted different regions of the same set of transcripts. This control allows us to assess the maximum proportion of significant sex-by-probe or line-by-probe interactions expected in the absence of differential transcript production . Of the 1,321 control probe sets, 129 (9.77%) showed significant interactions - a proportion that is well short of the 22% found for probes targeting distinct sets of transcripts. This suggests that although intrinsic probe biases and/or mistakes in the annotation may have an effect, this effect is not sufficient to explain the observed variation in relative transcript abundance. We conclude that a large proportion of multi-transcript genes in the CG7441, Sxl, fru, and Nep4, which showed evidence of sex-specific expression in the microarray data, were used as positive controls, while Lsp1beta, which was not sex-biased on the array, was used as a negative control. In all cases, qPCR results were consistent with array results . We then designed two to three primer pairs for each of nine genes that are known to be alternatively spliced and that showed evidence of sex-specific splicing in microarray experiments: unc-13, mud, Jupiter, r, aret, CG4662, CG10899, garz, and Akap200. These primer pairs were designed to amplify either constitutive exon junctions, or alternative splice junctions that were present in non-overlapping sets of transcripts. We measured the cycle thresholds of amplification (CT) for each primer pair in males and females of the Oregon-R line, and tested whether these values showed significant sex-transcript interaction. Such interaction would indicate that different exons were produced in different amounts in males versus females, confirming the microarray results. We observed statistically significant differences in transcript ratios in males versus females for eight out of nine genes  with primers that flanked exon junctions. First we evaluated the ability of qPCR to detect sex-biased transcript abundance. The genes X chromosome than on the autosomes using a \u03c72 test. For single-transcript genes, 57% (840) of the X-linked genes showed a significant difference in gene expression among sexes or lines, compared to 54%  for the autosomal genes. This difference, while slight, is greater than expected by chance (P = 0.0260). In other words, X-linked genes are significantly more likely to show differences in gene expression than autosomal genes. We then tested whether male- and female-biased genes were distributed in the same proportions between the X chromosome and the autosomes. We identified 559 female-biased genes on the X chromosome and 2,466 on the autosomes, compared to 281 X-linked and 2,164 autosomal male-biased genes. Thus, 18.5% of all female-biased genes are located on the X chromosome, while for male-biased genes the corresponding number is only 11.5%. This difference is highly significant (P < 0.0001), demonstrating that the X chromosome is enriched for female-biased single transcript genes.We tested whether the genes that showed evidence of differences in gene expression were more likely to be located on the X-chromosomal and 616 autosomal genes that showed a significant difference in gene expression in either sex or line; these showed no statistical evidence for chromosomal bias (P = 0.9479). However, among genes that showed sex-biased transcript abundance, 78 X-linked and 304 autosomal genes were female-biased, compared to 38 X-linked and 312 autosomal genes that were male-biased. The proportions of female- and male-biased genes located on the X chromosome  were significantly different (P = 0.0004), demonstrating that the X chromosome is enriched for female-biased multi-transcript genes.The same comparisons were performed for multi-transcript genes. There were 116 X-linked than for autosomal genes. There were 28 X-linked and 177 autosomal genes that showed significant sex-specific transcription; this proportion was not significantly different from that expected given the relative abundance of genes on the X chromosome and the autosomes (P = 0.3221). The male/female bias in alternative transcript representation was also independent of chromosomal location (P = 0.3479).We also tested whether sex-specific production of alternative transcripts (significant sex-by-probe interaction in the ANOVA model for multiple transcripts) was more likely to be observed for To perform a quantitative analysis of alternative transcript expression, we have designed transcript-specific probes based solely on sequence clustering . Definitions based on biological constructs such as exon junctions impose design restrictions that may result in probes that cross-hybridize to multiple genes, or do not have optimal hybridization properties with their intended targets. In contrast, our approach allows us to select probe sequences that will hybridize only to single transcripts. Our analysis shows that such probes perform in a uniform and highly reproducible fashion Table . MoreoveDrosophila, but also in Caenorhabditis elegans [A very large fraction of the genome appears to be differentially expressed between males and females. In our experiments, 53% of all expressed genes  showed sex-biased expression. Other studies utilizing different microarray platforms produced very similar estimates ,37-42. I elegans -45 and i elegans . This pa elegans , whereas elegans .X chromosome than male-biased genes . Similar 'feminization' of the X chromosome has previously been observed in Drosophila [C. elegans [We find that more genes show female-biased than male-biased expression (55% versus 45%). This result is in agreement with some of the previous reports , althougosophila ,41 and C elegans ,45.modulo and CG33092, show significant sex differences that change depending on the line examined . In contrast, some earlier reports suggested that as much as 10% of the genome may show such sex-genotype interactions [OregonR and 2b and six recombinant offspring from these two parents. The extent of alternative transcript production among lines will only be clear as more lines are sampled.We found that only two genes, ractions ,38. ThisDrosophila, alternative splicing plays a prominent role in development, most notably by controlling sex determination [Drosophila genes, alternative splicing is regulated in a sex-, tissue-, and/or stage-specific manner, so that different subsets of proteins encoded by the locus are produced in different developmental contexts [lola transcription factor have different functional domains, and mutations affecting the different isoforms have distinct phenotypes [Drosophila tyrosine hydroxylase  is required for cuticle development, while a different transcript functions primarily in neurotransmission [Dscam, which may produce up to 38,016 splicing variants [A large proportion of multi-exon genes in animal genomes are alternatively spliced, with estimates ranging from 30% to over 90% -24. Altemination -55. In acontexts ,56-61. Aenotypes . Similarsmission . One dravariants ,64. Recevariants . Howevervariants  may finaDrosophila genome. Approximately 22% of multi-transcript genes showed significant evidence that alternative transcripts were present in different ratios in males versus females. Some of these results might be experimental artifacts due to technical differences between probes, or mistakes in the current gene annotation. To address this concern, we used identified multiple probes that were predicted to hybridize to the same target transcripts as controls. Significant interactions between sex and probe will provide an estimate of the maximum proportion of significant tests that might be due to differences among probes, or problems with annotation. We found this proportion to be less than 10%, suggesting that at least 12% of all genes that produce alternative transcripts do so in a sex-specific manner. qPCR with primer pairs flanking alternative exon junctions confirmed sex-biased splicing for eight out of nine tested genes, indicating that exon-specific microarray probes provide a reliable means of detecting variation in the relative abundance of alternative transcripts. As in the case of sex-biased transcription, we suspect that much of the sex-specific splicing may be accounted for by reproductive tissues, and that most differences between males and females are likely to be quantitative rather than qualitative. Despite these qualifications, the prevalence of sexual differences in the production of alternative transcripts may have important functional consequences, and needs to be investigated in greater detail.We used the new microarray platform to estimate the extent of sex-specific production of alternative transcripts in the Drosophila genome contains a number of RNA-binding proteins that function as splicing regulators in vivo [dsx and Sxl [pasilla and mub genes disrupts the splicing of specific exons in the para and Dscam transcripts [Drosophila genome (approximately 53% in this study). We hypothesize that sex-specific expression of splicing regulators contributes to the prevalence of sex-specific production of alternative transcripts observed in our experiments. One attractive use of the new microarray platform would be to jointly monitor the expression of splicing regulators and the alternative transcripts of their target loci in different developmental contexts  and in different lines.The  in vivo . Importa and Sxl -71, whilnscripts . Thus, inscripts  are exprD. melanogaster. Sequences of 18,187 transcripts, including 16,064 transcripts annotated in FlyBase [a priori information about the exon/intron structure of the genes was used during cluster alignment. The overall set of 4,958 clustered transcripts contained 2,720 common and 2,545 unique regions. For most transcript clusters, common and unique regions identified by sequence alignment correspond to constitutively and alternatively spliced exons, respectively. Some examples of this correspondence are shown in Figure Our goal was to design microarray probes capable of distinguishing alternative transcripts, as well as members of multi-gene families. In order to maximize probe specificity, we first examined sequence similarity among all known and predicted transcripts of  FlyBase  and 2,12 FlyBase , were ob FlyBase . We iden FlyBase  to ident FlyBase . SequencDrosophila genome annotation, and subjected to a final BLAT verification. In particular, probes that were designed for singletons or unique regions were checked to make sure they did not match any other transcripts, whereas probes that were designed to represent common regions were confirmed to match only the expected set of transcripts.For each singleton transcript, and each unique and common region of clustered transcripts, we designed at least one 60-mer oligonucleotide probe. For 1,929 common regions of sufficient length to support non-overlapping probes that fit our design criteria, we designed two probes per region. To select the probes, we examined all possible 60-mers for each of the target regions, and scored each candidate based on several criteria, including GC content, OligoArray 2.1 score , homopolThe resulting microarray design included 12,994 probes that targeted singleton transcripts Table . If the Drosophila probes. These probes were compared to the Drosophila genome sequence to verify that they had no sequence similarity to any D. melanogaster genes. Five of these negative controls were randomly chosen for printing, and each was placed on the microarray one hundred times. At the end of the design there were an additional 3 spots available upon which negative controls were placed, for a total of 503 negative control spots. These negative controls allow us to estimate the distribution of signal intensities for probes that fail to hybridize, and to make present/absent calls for each transcript.We used the human genome to design 20 negative control probes according to the same criteria as the Drosophila transcripts, 503 negative control spots, and 1,304 Agilent controls  are targeted by each probe. Other columns enumerate the number of transcripts predicted for that CG in the current annotation, the number of transcripts the particular probe matches, the number of probes for that CG in the current microarray design, and whether the probe aligns with the gene with which it was originally designed to align. In this last column, four different designations may be given: 'match' , 'mismatch' (a different CG than expected), 'extended' , and 'not found' (no matches to any transcripts in the current FlyBase). Since the initial design includes predicted but unconfirmed genes, we expect that some probes will not be found in the current database. Additionally, probes are categorized into one of the following groups: 'singletons' (one match per probe), 'gene families' (match to more than one CG number), 'alternative transcripts' (one CG number represented by multiple common and unique regions), and 'pseudo-clusters' (more than one probe representing a single transcript). If two or more probes in an alternative transcript or gene family hit the same target region in the current annotation file, these probes were considered part of a 'set'. Each such set can then be classified as 'global' , or 'local' .D. melanogaster: OregonR [2b [Experiments were conducted on flies from two standard laboratory strains of  OregonR  and 2b [gonR [2b , and sixgonR [2b . Each ofRNA was extracted from each sample using Trizol reagent  according to the manufacturer's instructions, and purified using RNAeasy Kit . RNA concentration was determined using NanoDrop Spectrophotometer , and the sample quality was examined using the Agilent 2100 Bioanalyzer . We used 500 ng of RNA from each sample for the microarray experiment.Fluorescent cRNA was synthesized using the Aglient low RNA input fluorescent linear amplification kit following the manufacturer's protocol. Briefly, first and second strand cDNA was synthesized from 500 ng total RNA using an oligo dT-promoter primer and reverse transcriptase. Next, cRNA was synthesized using a T7 RNA polymerase, which simultaneously incorporates cyanine 3- or cyanine 5-labeled CTP. Labeled RNA was cleaned using Qiagen RNeasy columns and cRNA yield was quantified on a NanoDrop ND-1000 spectrophotometer. We pooled 750 ng of each labeled sample and hybridized to the arrays following the manufacturer's protocol. Hybridizations were performed with males and females of the same line labeled in contrasting dyes and hybridized to the same chip. We analyzed four independent biological replicates for each line and sex combination. For two of these replicates, males were labeled with Cy3 and females with Cy5, whereas for the other two the dyes were reversed. No technical replicates were performed as reliability of the Agilent platform is, on average, above 90% . This design maximizes the ability to test for sex effects (NIH project 5R24GM065513), and ensures that effects of sex remain balanced in the event of chip failure.Microarray experiments were carried out at the Interdisciplinary Center for Biotechnology Research Microarray Core, University of Florida. Hybridization occurred for 17 hours at 60\u00b0C in accordance with the manufacturer's instructions, and arrays were scanned using an Agilent Microarray scanner. There were seven technical failures, which were unrelated to the platform, leaving 25 successful hybridizations. Additionally, Agilent reported a manufacturing error that affected 2,310 spots on each chip, including 150 of the 503 negative controls. The failed chips and defective spots were removed from further consideration.Images were analyzed using Imagene software version 6.0 at the Purdue University Genomics Database Facility. Spots were individually quantified, and the mean intensities and mean background signal corresponding to each spot were exported into .csv files. As with other chip analysis software, in Imagene, the units are a function of pixel intensity. Individual files were collated for analysis at the Purdue University Genomics Database Facility. Transcript abundance was estimated as the natural log of the spot mean minus the mean of the local background.All spots on the array were compared between pairs of biological replicates to determine the reproducibility of RNA labeling and hybridization. Weighted kappa values ranged from 0.754 to 0.906, with a median of 0.85  if they hybridized in 50% or more of the replicates of that treatment. Probes that were not detected in at least one treatment were considered uninformative, and not considered further. The 20,265 available spots represented three groups of probes: Agilent controls , negative hybridization controls (353 spots), and Drosophila probes . There were 13,874 Drosophila probes (74%) found to hybridize in at least one treatment, including 187 of the 311 suboptimal probes  in this case is a combination of line and sex, and there were a total of 16 treatments since we examined 2 sexes for each of 8 lines. The ANOVA modeling approach compares means among groups, and determines whether the means are significantly different given the observed level of variation. To test whether a particular effect was statistically significant, we used the FDR approach [tj). That is, we tested whether the means were different among any of the 16 line/sex combinations (treatments). If this test was significant at FDR = 0.05, we declared this gene significant and investigated further whether the differences were due to sex, line, or interaction between sex and line effects at a very strict FDR of 0.05/3. To determine whether the relative amounts of alternative transcripts differed among sexes or lines, we tested the interaction between probe set and treatment (tpjk) and, if it was significant at FDR = 0.05, we further examined whether this was due to interaction between probe and sex or probe and line effects. For cases where the main effect of probe set (pk) was significant, we compared the effect of 'global' probes to each 'local' probe. The multiple transcript model was also fitted for gene families.where approach , which iapproach -85 (an iapproach ). BrieflSignificant probe-by-sex or probe-by-line interactions might arise not only as a consequence of genetic variation in alternative transcript production, but also as an artifact of intrinsic differences between probes. In order to estimate the rate of such artifacts, we used the model above to examine sets of probes that were expected to hybridize to the same transcript or group of transcripts . For such sets of probes, their relative intensities should, in principle, be identical in all treatments, and thus no significant probe by treatment interactions should be observed. By measuring the actual proportion of the control probe sets for which probe by treatment interaction is significant, we can estimate the rate of putative false positives. However, it should be noted that the expected hybridization targets of the probe sets are defined based on the current annotation, and it is possible that some of the probes are in fact hybridizing to different transcripts or sets of transcripts. Thus, this approach will probably over-estimate the number of false positives.For genes that had a single informative transcript, the following ANOVA model for single transcripts was fitted for each transcript individually:Yijl = \u03bc + di + tj + \u03b5ijlijl is the transcript abundance for dye i, treatment j, and replicate l; \u03bc is the overall mean of the transcript abundance for that transcript; d is the dye effect; and \u03b5 is the error. As above, a treatment (t) in this case is a combination of line and sex, and there were a total of 16 treatments since we examined two sexes for each of 8 lines. [Where Y8 lines. -92. SignOregon-R line as described above. For each sex, we used three biological replicates of four individuals each. To correct for differences in transcript abundance between sexes, samples were equalized by evaporation and resuspension in DEPC-treated water (DEPC: Diethyl pyrocarbonate). DNase I digestion  was carried out for 30 minutes at 37\u00b0C. Reverse transcription was performed on 5 \u03bcg of total RNA using oligo(dT)16, as described by the manufacturer . qPCR was performed on 100 ng of cDNA product in a total volume of 25 \u03bcl using TaqMan PCR Mix (Applied Biosystems). Primers for qPCR were designed to amplify either constitutive or alternative exon junctions of specific transcripts listed in Additional file 6. PCR amplification was detected using SYBR\u00ae Green I dye chemistry and ABI Prism 7900 Real Time PCR system (Applied Biosystems). CTs were determined using the AB7900 system SDS software and defined as the fluorescence intensity significantly above background during the exponential phase of amplification for all reactions. For each gene, CT values were analyzed using the ANOVA model:Total RNA was isolated from whole virgin adults of the Yijk = \u03bc + si + pj + spij + \u03b5ijkYij is cycle count for the ith sex and jth transcript for replicate k; \u03bc is the overall mean for that gene and \u03b5 is the random error. Specifically, we tested whether the sex by transcript interaction effect was significant at a nominal level of 0.05.where All programs developed during this work  as well a priori expectations of sex-biased expression. P values obtained from the ANOVA are given with the notation p<effect>. The CG number is given in actuals and the model used for analysis (Single transcript/multiple transcript) is given in the final column. P value for the sex by probe set interaction; and the list of transcripts targeted by each probe. See text for further details. P values of the likelihood ratio test (LRT) for a significant probe-sex interaction are also given. Note that for genes where only one transcript was tested, the test of the interaction between transcript and sex is not applicable (NA).The following additional data are available with the online version of this paper. a priori expectations of sex-biased expressionMicroarray results for several sets of genes for which we had Click here for fileActual_set_id is the unique identifier for each probe that hybridizes to the same set of transcripts, and actuals_cluster_id is a unique identifier that groups probes based upon CG number. Probeuid is the unique identifier for that probe sequence.Click here for fileP values obtained from the ANOVA are given with the notation p<effect>. The CG number is given in actuals and the model used for analysis (Single transcript/multiple transcript) is given in the final column.The Click here for fileResults of analysis based upon the probe level, as well as annotations from FlyBaseClick here for fileP value for the sex by probe set interaction; and the list of transcripts targeted by each probe. See text for further details.The columns are, in order: probe ID; gene name; whether hybridization signal detected by that probe is greater in males or females; log-transformed female/male expression ratio for each probe; probe set ID; class of probe ; Click here for fileP values of the Likelihood Ratio Test for a significant probe-sex interaction are also given. Note that for genes where only one transcript was tested, the test of the interaction between transcript and sex is not applicable (NA).We give the probe sequences used, all qPCR results as well as the original array results to facilitate comparison. The Click here for file"}
+{"text": "This BXH.ApoE\u2212/\u2212 population recapitulates several \u201cmetabolic syndrome\u201d phenotypes. The cross consists of 334 animals of both sexes, allowing us to specifically test for the dependence of linkage on sex. We detected several thousand liver gene expression quantitative trait loci, a significant proportion of which are sex-biased. We used these analyses to dissect the genetics of gonadal fat mass, a complex trait with sex-specific regulation. We present evidence for a remarkably high degree of sex-dependence on both the cis and trans regulation of gene expression. We demonstrate how these analyses can be applied to the study of the genetics underlying gonadal fat mass, a complex trait showing significantly female-biased heritability. These data have implications on the potential effects of sex on the genetic regulation of other complex traits.The integration of expression profiling with linkage analysis has increasingly been used to identify genes underlying complex phenotypes. The effects of gender on the regulation of many physiological traits are well documented; however, \u201cgenetical genomic\u201d analyses have not yet addressed the degree to which their conclusions are affected by sex. We constructed and densely genotyped a large F2 intercross derived from the inbred mouse strains C57BL/6J and C3H/HeJ on an apolipoprotein E null  for a measure of gonadal fat mass and for expression of transcripts in the liver. The results are used to explore the relationship between genetic variation, sexual differentiation, and obesity in the mouse model. Using over 300 intercross progeny of two inbred mouse strains, five loci in the genome were found to be highly correlated with abdominal fat mass. Four of the five loci exhibited opposite effects on obesity in the two sexes, a phenomenon known as sexual antagonism. To identify candidate genes that may be involved in obesity through their expression in the liver, global gene expression analysis was employed using microarrays. Many of these expression QTLs also show sex-specific effects on transcription. A hotspot for  Females and males share nearly identical genetic information, but vary widely with respect to disease susceptibility ,2. ApartWe have utilized the mouse as a model system to study the genetics of metabolic and vascular diseases \u20135. Rathe\u2212/\u2212) background. The BXH.ApoE\u2212/\u2212 population was designed to recapitulate several of the phenotypes associated with the so-called metabolic syndrome. The cross consists of 334 animals of both sexes, allowing us to specifically test for the dependence of QTLs on sex. We detected several thousand gene expression QTLs (eQTLs), a significant proportion of which were sex-biased. We used these analyses to dissect the genetic regulation of the gonadal fat mass trait and to identify genes associated with the trait.Here we report the application of this integrated approach to study the significance of sex on the genetic determinants of obesity and the associated regulation of liver gene expression in an F2 intercross derived from the inbred strains C57BL/6J (B6) and C3H/HeJ (C3H) on an apolipoprotein E null (ApoE\u2212/\u2212 and C3H.ApoE\u2212/\u2212 parents and the F2 BXH.ApoE\u2212/\u2212 generation on a Western diet are summarized in p < 10\u22124) and in the parental C3H.ApoE\u2212/\u2212 (p < 0.05), but not in B6.ApoE\u2212/\u2212 mice. Gonadal fat mass was the fat pad collection that represented the most animals and the most accurate collections and was thus chosen for further analysis. Broad sense heritability (h2) calculated as /\u03c32Total for the gonadal fat mass trait was 54% for females and 36% for males, which is in close agreement with previous reports [Characteristics of the B6.ApoE reports ,19 and dp < 1 \u00d7 10\u22123) and a significant threshold of 4.3 . QTL models incorporating only the sex*add interaction term in addition to the additive terms have one extra degree of freedom that leads to a corresponding increase in the LOD score thresholds to 3.5 (suggestive) and 4.9 (significant) for the 0.001 and 0.00005 p-value thresholds, respectively. QTL models incorporating both sex*add and sex*dom interaction terms possess two extra degrees of freedom, with a corresponding increase in LOD score thresholds to 4.0 (suggestive) and 5.4 (significant) for the 0.001 and 0.00005 p-value thresholds, respectively.A total of 334 F2 mice were genotyped at an average 1.5 cM density using 1,032 single nucleotide polymorphisms (SNPs) spanning all 19 autosomes. QTL analysis for several clinical traits , including the unadjusted raw values for gonadal fat mass, was performed using a single marker regression approach . In order to test specifically for sex effects of linkage, we included additive, dominant, sex, sex-additive, and sex-dominant parameters in our calculations see . A stepwOne suggestive (Chromosome 1) and four significant  cQTLs for the gonadal fat mass trait were identified . Four oup = 2 \u00d7 10\u22124); males analyzed alone did not demonstrate evidence for linkage. However, using all 334 animals and adding the interaction terms to the QTL model significantly improved sensitivity, and a cQTL with a maximum LOD of 7.56 (p = 1.7 \u00d7 10\u22126) was realized.Results from the various regression models used to determine linkage for the Chromosome 5 cQTL are depicted in R2 = 0.091 in females versus R2 = 0.046 in males), confirming the minimal sex specificity of this cQTL. The cQTL on Chromosome 19 showed a sex-specific effect in females, with no effect in males.Given the improved detection of four of the five cQTLs when sex-additive and sex-dominant interaction terms were considered, we hypothesized that the main genotype effect of these cQTLs on the gonadal fat mass trait would differ between the sexes . Indeed,Overall, all five cQTLs for gonadal fat mass were biased toward a larger effect in females. Assuming purely additive effects of each genotype, these cQTLs account for approximately 42% of the variation in female F2 mice and 13% in males, consistent with the narrow sense heritability estimates for this trait and again demonstrating significant differences in the regulation and heritability of the gonadal fat trait between the sexes.10 ratio (mlratio) of an individual experiment relative to a pool composed of 150 mice randomly selected from the F2 population [p < 0.05 in 10% or more of animals) relative to the pool is depicted in Livers from 312 F2 animals  were profiled using oligonucleotide microarrays manufactured by Agilent Technologies , which included probes for 23,574 mouse transcripts. Individual transcript intensities were corrected for experimental variation and normalized and are reported as the mean logpulation . Each mep < 5 \u00d7 10\u22125, genome-wide p < 0.05, based on a single trait), the FDR was estimated at 3.4% for the standard QTL model not accounting for any sex terms, 3.1% for the QTL model accounting for additive sex effects, and 3.2% for the QTL model accounting for additive sex effects and allowing for the sex interaction terms to enter the model. A list of all detected suggestive (p < 1 \u00d7 10\u22123) and significant eQTLs (p < 5 \u00d7 10\u22125) detected in the BXH.ApoE\u2212/\u2212 intercross is provided in The expression values of the 23,574 transcripts were treated as quantitative traits and fitted to the same linear regression models used to compute LOD scores for clinical traits (eQTLs). The FDR at each threshold was determined by permuting the data 100 times and taking the mean number of QTLs detected over all of the permuted datasets at a given threshold, and dividing this count by the number of QTLs detected at the same threshold in the observed data. At the threshold for significant linkage  of the corresponding gene, likely representing eQTLs regulated by cis-acting variation within the gene itself. Of the 6,676 significant eQTLs, 1,166 (17%) demonstrated a sex bias and were subsequently significantly improved with the addition of the sex-additive and sex-dominant terms.Characteristics of the eQTLs at different significance levels are summarized in p < 5 \u00d7 10\u22125) across the genome in 2 cM bins is shown in trans, and these eQTL hotspots consist primarily of trans-acting effects on transcriptional variation. Distribution of the 1,166 eQTLs with significant sex effects, of which 852 (73%) are trans, showed enrichment on Chromosome 5 at approximately 49 cM (p = 8.7 \u00d7 10\u221225 after Bonferroni correction). At this locus there were 250 eQTLs, 140 of which exhibited genotype\u2013sex interactions.The distribution of all 6,676 significant eQTLs . These data demonstrate the profound effects of sex on the genetic regulation of gene expression.At increasing thresholds for linkage, a higher fraction of detected eQTLs were s-acting C. The ind before ,15 and cresholds C. Furthepression D, and sicis-eQTLs with significant \u201csex-additive\u201d and \u201csex-dominant\u201d interactions would be potential candidate genes for our trait. Of the 2,118 significant cis-eQTLs, 304 (14%) were improved by the sex-interaction terms. Cis-eQTLs overlapping the confidence interval for the fat mass cQTL are candidate genes for the trait. Those genes with significant sex interactions receive increased consideration as potential candidates . Of the 4,613 genes correlated with gonadal fat mass, 1,130 generate 1,478 significant eQTLs B. The cotrans-eQTLs coincident with the fat mass cQTL cannot be candidates directly responsible for the trait, as they are physically located elsewhere in the genome. However, they are potentially involved in the pathway(s) leading from the causative gene to the expression of the fat mass trait . All of the five fat mass cQTLs have colocalizing trans-eQTLs for correlated genes. However, for a trait such as fat mass that is regulated by multiple tissues and organs, it is unlikely that all five fat mass cQTLs are primarily driven by liver gene expression. As an approach to this problem, we hypothesized that those cQTLs that are most closely associated with liver gene expression would show an overrepresentation of colocalized eQTL for correlated genes, while those loci primarily controlled by other tissues would not have shown this pattern.Genes that show significant correlation with gonadal fat mass and have p < 0.001 for enrichment along the genome in overlapping 2-cM bins against the distribution of all liver eQTLs  differs between males and females for the majority of the transcripts (as in p < 0.001 by \u03c72). This locus corresponds to one of the four sex-biased cQTLs for gonadal fat mass reported in this study, and the significant sex specificity of both the cis and trans genetic regulation of liver genes correlated with fat mass supports the functional significance of this locus in this organ.The effect of the ts as in A, and fots as in B, femaleIn this study, we described a large, densely mapped, segregating F2 mouse population designed to study the genetic regulation of several traits associated with the so-called metabolic syndrome. Several groups, including ours, have reported the advantage of combining traditional genetics with genome-wide gene expression analysis for the dissection of complex traits. This study improved on past models by including over 300 animals (three times the size of previous studies) of both sexes, allowing for the incorporation of sex-specific effects on underlying genetic regulation.Given the known dichotomy between females and males in the susceptibility and control of obesity, this study was designed to sufficiently power the detection of significant QTLs for this and other traits with sex-dependent effects. Note, however, that these effects can extend to traits without overall mean differences between the sexes. Previous studies have described the advantages of performing QTL analysis both with and without sex as an interactive covariate \u201325. Anal\u2212/\u2212 background and the high-fat Western diet. This background possesses several advantages, such as allowing the modeling of human-like disease states. The predominantly female-driven effects of the five cQTLs likely reflect the significant effect of differential gonadal hormone secretions on the genetic regulation of this complex trait.Accordingly, we detected five cQTLs for the gonadal fat mass trait on Chromosomes 1, 3, 5, 11, and 19. The detection of all five cQTLs was \u201cdriven\u201d by the larger effect in females, with significant improvement by the incorporation of sex*additive and sex*dominant parameters. QTLs associated with obesity, gonadal fat, and abdominal fat have been reported before overlapping with cQTLs on Chromosomes 1 \u201328, 5 2,29, and cis-acting variations affecting their transcription are potential candidate genes for the trait. At a single trait, genome-wide significance level of 0.05, we detected 6,676 eQTLs representing 4,998 genes, of which 2,118 were cis-acting. At increased thresholds, the proportion of cis-eQTLs increased, which is in good agreement with previous studies [cis-acting variations affecting transcription. Of all 6,676 significant eQTLs, 1,166 possessed significant sex interactions. Of these, 304 were cis and 852 were trans, suggesting that only a minority of the sex-specific effects on the regulation of gene expression occur through polymorphisms within the gene itself. Rather, underlying genetic regulation of most transcripts is the result of interactions between trans loci and sex-specific factors . As with cQTLs, sex bias in the predominantly trans genetic regulation of gene expression is likely secondary to different sex hormone profiles.The identification of genes underlying cQTLs remains a challenge. The widespread availability of genome-wide expression analysis has begun to address this by providing a snapshot of transcription in relevant organs and thus providing initial information for which genes can differentiate a given trait. Furthermore, by treating transcript levels as quantitative traits, we can map the genetic regulation underlying differential gene expression (eQTLs). Those eQTLs that have  studies  and likecis-eQTLs (p < 5 \u00d7 10\u22125) largely represent true positives [cis-eQTLs presented in cis-eQTLs with significant sex*additive and sex*dominant effects should receive priority consideration. The use of eQTLs to dissect cQTLs is a method still in its infancy, with uncertain efficacy and applicability. Nevertheless, application of this analysis to this dataset provides some tantalizingly attractive candidate genes.Recently, using a similar dataset, our group demonstrated that significant ositives  and are cis-eQTLs. However, it is not strictly necessary for candidate genes to have evidence of such linkage: polymorphisms underlying a trait cQTL can affect gene function or posttranslational modifications. Nevertheless, several phenotypes are known to be regulated, at least partly, at the level of transcription or mRNA stability, which is exactly what our methods are designed to detect. A separate problem is that organ-specific gene expression differences may preclude one from detecting the relevant causative gene if the tissue arrayed is not the tissue where the control is exerted. This is particularly relevant for a trait such as adipose tissue mass, which is controlled by multiple tissues. We propose that analysis of correlated genes can provide guidance as discussed below.One shortcoming of this approach, however, is that candidate genes are limited to those whose transcript expression levels vary in association with a nearby polymorphism that differs between the parental strains\u2014in other words, genes with significant and detectable candidate genes underlying the trait cQTLs, we fitted linear models to assess the degree of association between transcripts and gonadal fat mass. As with QTLs, sex-specific correlations were modeled. At an FDR of 1%, 4,613 genes were found to be significantly correlated with gonadal fat mass, of which 4,254 (98%) showed sex-biased correlation. As indicated in cis-eQTLs are also significantly correlated with the trait and are even further prioritized as candidate genes.In an effort to identify genes associated with the fat mass trait, but not necessarily cis-eQTLs relative to trans [trans-eQTLs and the nature of the underlying polymorphisms associated with them. Furthermore, the eQTL hotspots reported in this and previous studies [trans-eQTLs. This localization suggests some functional significance to these regions. Of the 4,613 genes correlated with gonadal fat mass, 1,130 generate 1,478 significant eQTLs, of which 1,023 (69%) are trans-acting. These eQTLs are significantly enriched at one locus (Chromosome 19). Interestingly, this hotspot was coincident with a cQTL associated with fat mass reported in this study. Since these transcripts represent those significantly correlated with gonadal fat mass, the localization of their eQTLs to these regions strongly supports the notion that the genes with trans-eQTLs represent downstream targets of candidate regulatory genes located at the position of significant linkage. This means that the genes may be causal but downstream of the gene responsible for the cQTLs, or they may be reacting to the increased gonadal fat mass and associated metabolic changes. These data also suggest that identifying such loci that show overrepresentation of highly correlated genes is a means to identify which of the trait cQTLs are more likely controlled by the tissue arrayed. As expected, the Chromosome 19 locus was enriched for trans-eQTLs with substantially greater effects in females. Functional and promoter analysis of genes with a common trans-eQTL may prove enlightening. Furthermore, gene expression network construction and analysis may be improved by the incorporation of experimentally demonstrated cis versus trans regulation.Thus far, studies that have examined the \u201cgenetics of gene expression\u201d are in good agreement regarding the increased power to detect to trans ,15,16,31 studies ,10 largeThe integration of traditional genetics with genome-wide expression analysis was first proposed by Jansen and Nap 4 y ago . Advancetrans to a cQTL region can identify relative tissue contributions to the genetic regulation of a complex trait. We anticipate that the application of these and similar methods would significantly improve the elucidation of the genetic regulation underlying complex phenotypes.We reported here on the initial genetic and genomic analysis of an F2 intercross population designed to recapitulate several traits associated with human metabolic syndrome. Using 334 mice of both sexes genotyped at high density, this is the largest study of its kind to date designed, and it is strongly powered to detect subtle effects of genetic regulation and sex specificity. We identified five cQTLs for the gonadal fat mass trait, all with greater effects in females. We also detected several thousand significant liver eQTLs, a significant fraction of which are sex-biased, demonstrating how meaningful effects of sex on gene expression extend beyond overall mean differences. We demonstrated the application of linkage and correlation methods to identify candidate genes. Finally, we showed that localization of a subset of liver genes linked in \u2212/\u2212 (B6.ApoE\u2212/\u2212) were purchased from Jackson Laboratory . C3H/HeJ ApoE\u2212/\u2212 (C3H.ApoE\u2212/\u2212) were generated by backcrossing B6.ApoE\u2212/\u2212 to C3H for ten generations. F1 mice were generated from reciprocal intercrossing between B6.ApoE\u2212/\u2212 and C3H.ApoE\u2212/\u2212, and F2 mice were subsequently bred by intercrossing F1 mice. A total of 334 mice  were produced. All mice were fed Purina Chow containing 4% fat until 8 wk of age and then transferred to a \u201cWestern\u201d diet containing 42% fat and 0.15% cholesterol for 16 wk. Mice were sacrificed at 24 wk of age. At death, livers were immediately collected and flash-frozen in liquid N2, and gonadal fat pads were extracted and weighed.C57BL/6J ApoERNA preparation and array hybridizations were performed at Rosetta Inpharmatics . The custom ink-jet microarrays used in this study  contMouse livers were homogenized and total RNA extracted using TRIzol reagent  according to manufacturer's protocol. Three micrograms of total RNA was reverse transcribed and labeled with either Cy3 or Cy5 fluorochrome. Purified Cy3 or Cy5 complementary RNA was hybridized to at least two microarray slides with fluor reversal for 24 h in a hybridization chamber, washed, and scanned using a laser confocal scanner. Arrays were quantified on the basis of spot intensity relative to background, adjusted for experimental variation between arrays using average intensity over multiple channels, and fitted to an error model to determine significance (type I error). Gene expression is reported as the mlratio relative to the pool derived from 150 mice randomly selected from the F2 population. For subsequent analyses, mlratio data are assumed to be normally distributed, a valid assumption as previously demonstrated ,30. The y. The linear model that relates variation in y to QTLs and other covariates  is given by the general formGenomic DNA was isolated from kidney by phenol-chloroform extraction. An examination of existing databases identified over 1,300 SNPs that showed variation between the B6 and C3H strains, and a complete linkage map for all 19 autosomes was constructed using 1,032 of these SNPs at an average density of 1.5 cM. Genotyping was conducted by ParAllele  using the molecular-inversion probe (MIB) multiplex technique . Testinge the residual error. Linkage was computed for over 20 clinical traits as well as 23,574 liver transcripts. Standard genome scans calculate linkage by comparing the linear modelwhere \u03bc is the trait mean, X\u2032 a vector of covariates, \u03b2 being the associated vector of regression coefficients, and to the null model1 and \u03b22 are the regression coefficients of the additive and dominant parameters, respectively. The LOD score represents the difference in the log10 of the likelihood of the above two equations, where the individual model likelihoods are maximized with respect to the model parameters, given the marker genotype and phenotype data. If a trait y differs on average between the two sexes but the QTL has the same effect in both males and females, we can model this interaction by including sex as an additive covariate in the above models, resulting in the new modelwhere \u03b2which is then compared to the null model3 is the regression coefficient of the sex parameter. The effect of a QTL may be dependent on the state of a covariate; for instance, a QTL may have an effect specific to one sex, or may have opposite effects in the two sexes. This interaction can be modeled using a full model, which accounts for all additive covariates, as well as interactions between the covariates:where \u03b2which is compared to the above null model .\u22123 and 5 \u00d7 10\u22125 equivalent to LOD scores of 4.0 (suggestive linkage) and 5.4 , respectively. As a result, if there are no significant sex-additive or sex-dominant interactions, the full model will actually reduce power to detect linkage. To minimize the loss in power of fitting the full model when the sex-interaction terms are not significant, we employed a model selection procedure that introduces sex-interaction terms only if they add significantly to the overall QTL model.The full model  allows up < 0.001). That is, the data are fitted to The model selection procedure makes use of forward stepwise regression techniques to determine whether it is beneficial to include the sex-interaction terms, conditional on realizing a significant additive effect  and 1,159 (45% increase) QTLs were detected for Equations For each 23,574 oligonucleotides represented on the array, we computed a linear regression analysis to test for a correlation between the trait \u201cgonadal fat mass\u201d and each transcript. Similar to our method of calculating linkage, we employed a stepwise linear regression procedure using equations of the formorcompared to the null model0 is the intercept, and \u03b21, \u03b22, and \u03b23 represent the regression coefficients of their respective terms. As before, the parameter \u201csex*gene\u201d is only retained if it significantly improves the fit of the model. The p-value threshold for significant correlation is calculated by an F test, which compares the appropriate model . Loci with p < 6.5 \u00d7 10\u22125 by Fisher exact test were considered significantly enriched for eQTLs correlated with gonadal fat mass.Genes correlated with the gonadal fat mass trait generated several significant eQTLs. In order to determine if eQTLs generated by these genes were enriched in any locus or if they were distributed randomly, we compared the distribution of these eQTLs against the distribution of all liver eQTLs in overlapping 6-cM bins across the genome using the Fisher exact test. This test is based on exact probabilities from a specific distribution (hypergeometric distribution). Figure S1(21 KB DOC)Click here for additional data file.Table S1(3.7 MB XLS)Click here for additional data file.Table S2(716 KB XLS)Click here for additional data file."}
+{"text": "Staphylococcus aureus.DNA microarray technology is widely used to determine the expression levels of thousands of genes in a single experiment, for a broad range of organisms. Optimal design of immobilized nucleic acids has a direct impact on the reliability of microarray results. However, despite small genome size and complexity, prokaryotic organisms are not frequently studied to validate selected bioinformatics approaches. Relying on parameters shown to affect the hybridization of nucleic acids, we designed freely available software and validated experimentally its performance on the bacterial pathogen Staphylococcus aureus. BLAST analysis of the final selection of 5,427 probes yielded >97%, 93%, and 81% of Staphylococcus aureus genome coverage in strains N315, Mu50, and COL, respectively. A manufactured oligoarray including a subset of control Escherichia coli probes was validated for applications in the fields of comparative genomics and molecular epidemiology, mapping of deletion mutations and transcription profiling.We describe an efficient procedure for selecting 40\u201360 mer oligonucleotide probes combining optimal thermodynamic properties with high target specificity, suitable for genomic studies of microbial species. The algorithm for filtering probes from extensive oligonucleotides libraries fitting standard thermodynamic criteria includes positional information of predicted target-probe binding regions. This algorithm efficiently selected probes recognizing homologous gene targets across three different sequenced genomes of This generic chip-design process merging sequence information from several related genomes improves genome coverage even in conserved regions. Such massively parallel systems offer unprecedented opportunities for basic research and diagnostic applications, including gene sequencing , detectiet al ).Using a spreadsheet program, the probes were sorted by their ORF target names and their best thermodynamic criteria. Probes showing the best combination of thermodynamic criteria and strain coverage were selected for microarray manufacturing Fig. . In addiA compiled version of OliCheck compatible with Windows 2000 or XP and written in the Delphi programming language  is freely downloadable for non-profit use at Genomic Research Laboratory website .Three probes set of 60-mer probes with homogenous thermodynamic criteria (Tm = 60\u00b0C) were generated using default parameters for N315 genome by: i) ArrayDesigner ii) ArrayOligoSelector for all candidate probes, iii) ArrayOligoSelector for the best candidate probes (one per ORF). The output lists generated by ArrayOligoSelector were reformatted to match OliCheck input file format. The list of probes generated by either software was further processed by OliCheck for cross-homology filtering. The sets of probes selected by each method were further compared for homology using BLAST. Alignment showing E-value <1E-20 were considered as homologous.in situ synthesis of 8,455 long oligonucleotide probes (Agilent). It consists of 5,427 S. aureus and 2,873 E. coli specific probes, together with A. thaliana control probes for spiked controls.The StaphChip microarray was manufactured by S. aureus strain was grown overnight in 2 ml Mueller-Hinton broth (MHB) and total DNA was extracted and purified using DNeasy columns (QIagen) following manufacturer's instructions. DNA purity and concentration were assayed by spectrophotometer. 2 \u03bcg DNA were labelled by the Klenow fragment of DNA polymerase I  with Cyanine-3 or Cyanine-5 coupled dCTP (NEN) for 2 hours at 37\u00b0C, then stopped by the addition of 5 \u03bcl 0.5 M EDTA. Labelled DNA was purified on QiaQuick columns (Qiagen).For comparative genome hybridization, each S. aureus RNA were spiked with increasing amounts of different Arabidopsis thaliana mRNAs , used as external calibrators. The RNA mixture was labelled by Cy-3 dCTP or Cy-5 dCTP, using the SuperScript II (Invitrogen) following manufacturer instructions. Labelled cDNA was then purified onto QiaQuick columns.For gene expression analysis, total RNA was extracted from 2 ml exponential or overnight cultures using the Rneasy kit (Qiagen) as previously described . BatchesS. aureus strain was labelled with Cy3 and co-hybridized with equivalent amounts of Cy5-labelled genomic DNA pooled from N315, Mu50 and COL [Unless specified, equivalent amounts of cDNA (or genomic DNA) labelled with Cyanine-3 or Cyanine-5, were diluted in 250 \u03bcl Agilent hybridization buffer, and hybridized at a temperature of 60\u00b0C for 17 hours in a dedicated hybridization oven (Robbins Scientific). For comparative genome hybridization, genomic DNA from each individual  and COL . Slides For gene expression analysis, saturated spots were excluded from subsequent analysis. Local background-subtracted signals were corrected for unequal dye incorporation or unequal load of labelled product. The algorithm consisted of a rank consistency filter and a curve fit using the default LOWESS  method. Spots showing a reference signal lower than background plus two standard deviations were also excluded from subsequent analyses.10 ratios and analyzed by two-way clustering using GeneSpring 6.1 (SiliconGenetics).For comparative genome hybridization, local background-subtracted data were expressed as LogThe expression of 18 genes involved in metabolic pathways was quantitatively assayed using by 1 step RT-qPCR using a SDS7700 . Primers and probes were identified by scanning each gene sequence using the software Primer Express 2.0(Applied Biosystems). All identified sequences were further aligned on the whole genome of sequenced strains to ensure gene specificity and conservation of the target sequence between strains. Optimal concentration of primers and Taqman probes , determined accordingly to manufacturer's instructions were 200 nM and 100 nM per reaction, respectively. Primers and probes were mixed in Platinum qRT-PCR Thermoscript kit (Invitrogen) with 0.4 ng of total purified RNA. Fold changes were calculated after normalization with the expression level of the 16s rRNA gene as previously described .YC performed Olicheck implementation, designed microarray and wrote the manuscript. BG performed Olicheck implementation, designed microarray and helped writing the manuscript. PF participated in the design of the study, designed experiment protocols, performed microarray analysis. MB contributed to design experiment protocols and performed microarray experiments. AR has been involved in the CGH project. PV has been involved in drafting the article and revising it critically for intellectual content. WS has made substantial contributions to conception and design. JS initiated the study and helped writing the manuscript. All authors read and approved the final manuscript."}
+{"text": "Although a large proportion of human transcription occurs outside the boundaries of known genes, the functional significance of this transcription remains unknown. We have compared the expression patterns of known genes as well as intergenic transcripts within the ENCODE regions between humans and chimpanzees in brain, heart, testis, and lymphoblastoid cell lines. We find that intergenic transcripts show patterns of tissue-specific conservation of their expression, which are comparable to exonic transcripts of known genes. This suggests that intergenic transcripts are subject to functional constraints that restrict their rate of evolutionary change as well as putative positive selection to an extent comparable to that of classical protein-coding genes. In brain and testis, we find that part of this intergenic transcription is caused by widespread use of alternative promoters. Further, we find that about half of the expression differences between humans and chimpanzees are due to intergenic transcripts. In order to convert the genetic information encoded in an organism's genomic sequence into the functional features, the genomic sequence must be transcribed. According to the current genome annotation, the human genome encodes 20,000\u201325,000 protein-coding transcripts and a smaller number of non-coding transcripts. There is, however, a growing body of evidence indicating that a much greater proportion of the human genome is transcribed than is accounted for by the existing annotation. Much of this evidence has been found using tiling arrays, microarrays that enable the measurement of transcription regardless of existing annotation. Although some have suggested that these transcripts represent previously unidentified functional RNAs as well as extensions of known genes, the extent of their functionality remains unknown. In this study, Khaitovich et al. assess the functionality of these novel transcripts by testing the extent to which their expression is conserved between humans and chimpanzees in different tissues. The results suggest that, surprisingly, the expression of both known and novel transcripts was affected by the same functional constraints during human and chimpanzee evolution. RNA transcription is the first step in transferring the information encoded in a genome sequence into the phenotypic features of an organism. Naturally, much effort has been spent determining which regions of the human genome are transcribed and for what biological purpose. Until recently, these efforts were largely restricted to either computational gene prediction or alignment of sequenced cDNAs and ESTs to the genomic sequence. Based on information collected using these methods, the human genome annotation is converging on a set of 20,000\u201325,000 protein-coding genes and a smaller set of non-coding RNAs. However, there is a growing body of evidence acquired using a variety of new approaches that indicates that much more transcription occurs than is accounted for by the existing annotation \u20133.Tiling arrays are a powerful new tool for detecting transcription in an unbiased manner ,4\u20136. On In general, intergenic transcripts tend to be expressed at low levels, sometimes at or below the detection limit of Northern blot or RT-PCR techniques ,9,12. InThis lack of sequence conservation has led to the suggestion that intergenic transcripts represent the products of stochastic and unproductive activity of the RNA transcription machinery and are consequently degraded . HoweverWe have analyzed the evolutionary conservation of expression patterns between transcripts of known genes, as well as transcripts derived from intergenic regions in humans and chimpanzees, by measuring transcription in a strand-specific manner from both DNA strands in the 44 genomic regions studied by the ENCODE consortium covering approximately 1% of the human genome (to which we will refer as the ENCODE regions) in four tissues  from five humans and five chimpanzees, using tiling arrays based on the human genome sequence , a greater proportion of exonic probes and a lesser proportion of intergenic probes are classified as expressed. Still, in each tissue we find on average 2.4 times more expressed intergenic than expressed exonic probes . It shouWe next investigated whether the same probes are expressed in humans and chimpanzees in each tissue. This is important, because if intergenic expression is stochastic in nature, we might expect to find similar amounts of transcription but little overlap between the intergenic probes expressed in two species. Since the reliability of expression detection depends greatly on signal intensity, and exonic and intergenic probes have different signal intensity distributions, we limited our analysis to exonic and intergenic probes chosen to have the same distributions of signal intensity . By compPrevious studies have indicated that genes expressed in different tissues experience different extents of negative and positive selection on both DNA sequence and gene expression levels ,20. On tWe then investigated whether differences in diversity and divergence can also be observed for intergenic transcripts. Since we assume that these differences in transcript expression observed among tissues arise due to differences in the extent of functional constraints and positive selection in these tissues, in the absence of any function, no such differences would be expected. Thus, if we assume as a null hypothesis that intergenic transcripts have no function and represent products of spurious activity of the RNA transcription machinery, we expect that their expression diversity and divergence patterns should be the same among the three tissues. We therefore tested whether this is the case by comparing intergenic and exonic probe expression patterns in the three tissues. In order to avoid artifacts caused by differences in technical measurement errors associated with different probe signal intensities, we compared exonic and intergenic probes having the same signal intensity distribution . HoweverA potential alternative explanation for the observed difference in intergenic expression patterns among tissues is cross-hybridization of transcripts of known genes to intergenic probes. We tested whether this may be the case by consecutively removing all probes that map to more than one location in the human genome with zero, one, two, three, four, or five mismatches, respectively. Although this procedure does not necessarily remove all probes that could cross-hybridize to transcripts of known genes, we would expect the difference between tissues seen in p < 10\u22126) between expressed intergenic probes and the nearest exon in all four tissues. This may indicate that some intergenic expression arises from as-yet unannotated extensions of known genes. In order to test if this could be the case, we calculated the correlation between the signal intensity of intergenic probes with the signal intensity of the nearest exon. Overall, we find no such correlation (p < 0.05), but not in heart (p = 0.39) nor in lymphoblastoid cell lines (p = 0.36). In both brain and testis, this positive correlation is seen for probes located upstream, but not downstream, of the nearest exon . We find that in each individual tissue, about half of the total number of differently expressed probes originate in intergenic regions  reagent according to the manufacturer's instructions and purified with MiniElute  kit following the manufacturer's instructions with no modification. All RNA samples used in this study were of high and comparable quality as gauged by the ratio of 28S to 18S ribosomal RNAs estimated using the Agilent 2100 Bioanalyzer system   by the same person (PK). Human and chimpanzee samples were matched with respect to sex and relative age . Total R States) . The totAll samples were processed, labeled, and hybridized to Affymetrix  ENCODE 0.1 FORWARD and REVERSE arrays following the Whole Transcript Sense Target Labeling Assay protocol , with few modifications. Namely: 2 \u03bcg of total RNA were used in rRNA reduction protocol instead of the recommended amount of 1\u03bcg; 2.5 times greater than the recommended total reaction volume was used in the cDNA synthesis step; cDNA cleanup was carried out following standard phenol-chloroform extraction protocol; the entire amount of purified cDNA was used for the cRNA synthesis step carried out with Ambion Megascript kit  according to the manufacturer's instructions for 16 h at 37 \u00b0C; and all RNA cleanup steps in the protocol were carried out with Qiagen MiniElute kit following the manufacturer's instructions with no modifications.Samples for the FORWARD and REVERSE version of arrays were processed, labeled, and hybridized independently.http://www.r-project.org). Prior to analysis we masked all oligonucleotide probes that are not 100% identical over their full length to both human (NCBI build 35) and chimpanzee (panTro1) genome sequences. This reduced the number of probes available for analysis from 755,455 to 503,459. We defined each probe as a pair of sequences, one perfectly matching the designated genomic sequence (PM) and another, with a transversion in the central position (MM).Affymetrix microarray image data were acquired using an Affymetrix 3000 scanner with the default settings. The intensity of individual probes was calculated using standard Affymetrix GeneChip Operating Software. Resulting raw probe intensity files were analyzed using R (http://www.bioconductor.org) as a difference between perfect PM and mismatch MM signal intensities.All arrays used in this study were normalized together using \u201cmas\u201d background correction and scaling normalization procedures based on all remaining 503,459 probes. Expression values for each probe were calculated using the \u201caffy\u201d Bioconductor software package (http://genome.imim.es/gencode) of the ENCODE consortium [http://hgdownload.cse.ucsc.edu/goldenPath/hg17/encode/database/). Using these annotations, we assigned array probes to genomic regions corresponding to known genes, putative genes, and pseudogenes. We omitted probes falling within pseudogenes from further analyses. This further reduced the number of probes from 503,459 present on each strand to 498,556 on the positive and 498,527 on the negative strand. For each DNA strand, the remaining probes were separated into those mapping within exons and introns of known genes located on the relevant strand and intergenic probes. Intergenic probes were defined as mapping outside of the exons and introns of known genes on both strands. This excluded from analysis all potential antisense transcripts corresponding to genomic regions of known genes. However, the labeling protocol we used is known to produce spurious second-strand transcripts at the first cDNA synthesis stage, which would then be classified as \u201cantisense transcripts\u201d [http://genome.imim.es/gencode) of the ENCODE consortium based on gene predictions as putative gene regions. However, excluding probes mapped within these regions did not affect any of our results  between the two nearest probes for all intergenic and exonic expressed probes. The distribution of distances expected by chance was calculated by 1,000 permutations of expressed probe assignments within exonic and intergenic regions A. The saThe proximity between expressed intergenic probes and known exons was tested within each of the 44 ENCODE regions by calculating the genomic distance between all expressed intergenic probes and the nearest annotated exon. Chance distribution was calculated by 1,000 permutations of expressed probe assignments within intergenic regions. The difference between the observed distance distributions for all regions was compared to that expected by chance using the Wilcoxon rank test.p < 0.05, corrected for the number of regions used in analysis). For the results presented in The correlation between expressed intergenic probes and the nearest expressed exon, based either on the absolute signal or on the signal intensity difference between humans and chimpanzees, was calculated as follows: First, we calculated the mean signal intensity or mean signal intensity difference for each GENCODE known or known + putative exon, using all probes located within the exon. For each tissue, we defined the expressed exons as those containing at least one expressed probe. Further, for each expressed intergenic probe within each of the 44 ENCODE regions, we determined the nearest expressed exon by either disregarding their relative positions, or by separately determining the nearest exons located 5\u2032 or 3\u2032 from the intergenic probe. We then determined the Spearman rank correlation between the absolute signals or between the signal intensity differences of all expressed intergenic probes and probes in the nearest exon within each of the 44 ENCODE regions. Regions containing less than 100 expressed exonic probes were excluded from the correlation analysis. For each tissue, the mean correlation coefficient was based on the correlation coefficients for all regions showing significant correlation , FDR in all tissues was less than 5%. Genomic coordinates of the differently expressed probes on the human genome (Build 35) are listed in Probes showing a significant expression difference between humans and chimpanzees were identified by applying the Student's http://www.affymetrix.com/support/downloads/library_files) to both the human and the chimp genomes. Oligonucleotide probes that matched both genomes perfectly were retained for the analysis while the probes with mismatches to either genome were masked.All probes on the ENCODE arrays were designed using the human genome sequence as a reference. To eliminate the effects of sequence divergence between humans and chimpanzees on the estimation of expression levels, we excluded from all analyses the array probes that do not match both the human (NCBI build 35) and the chimpanzee (panTro1) genome sequences with 100% identity over the entire probe length. Using BLAT , we aligAdditionally, to study the potential effects of cross-hybridization, we removed from the set of perfectly matching probes those probes that could be aligned to more than one genomic location with zero, one, two, three, four, or five mismatches.http://genome.ucsc.edu/goldenPath/help/phastCons.html). The seven species included in these alignments were: chimpanzee, dog, mouse, rat, chicken, zebrafish, and fugu. PhastCons uses maximum likelihood to fit a phylogenetic hidden Markov model to the sequence data of the different species. Each genome base is assigned a score reflecting the probability that the base position is in its most conserved state, according to the hidden Markov model. For each probe, the conservation score was calculated as a mean of all available PhastCons conservation scores for the bases within the probe. Probes with no available PhastCons conservation scores were removed from the analysis.DNA sequence conservation scores were based on the sequence conservation measures for each nucleotide position provided by the PhastCons conservation scores for eight-way multiple alignments between the human genome and genomes of the seven vertebrate species assemblies to the human genome build hg17, May 2004  primer for the first strand cDNA synthesis. To control for possible amplification from genomic DNA, we used as a negative control cDNA prepared without the addition of reverse transcriptase during synthesis. As a positive control, we used PCR primers for a GAPDH transcript. The primers for this transcript were placed in different exons resulting in a PCR product of 602 bp in length if genomic DNA was amplified and a PCR product of 285 bp in length if cDNA was amplified. PCR products were visualized on agarose gels using ethidium bromide staining. The signal intensity of the PCR bands was quantified using ImageQuant . The signal intensity from the negative control lane was subtracted from the products' signal intensities to correct for unspecific amplification and signal background. Further, we normalized the products' signal intensities using the signal intensity of GAPDH transcripts measured four times in the six individuals used in Q-PCRs in order to correct for differences in the total amount of cDNA derived from each individual. The primers' sequence, their calculated melting temperatures, their corresponding positions in the human genome, the product size, the melting temperature, the GC content, and the expected product length are shown in Figure S1x-axis shows the number of exonic and intergenic probes. Red indicates the proportion of probes we classified as expressed.The distributions of signal intensities of exonic (left) and intergenic (right) probes are shown for brain (A), lymphoblastoid cell line (B), heart (C), and testis (D). The signal intensity range presented in each panel covers 95% of all array probes with positive signal intensity. The (260 KB PDF)Click here for additional data file.Figure S2The distribution of expressed probes among exonic (blue), intronic (gray), and intergenic (red) regions is shown in four tissues in humans (upper row) and chimpanzees (lower row) for the probes corresponding to the positive (A) and the negative (B) DNA strand. The \u201cTotal\u201d indicates the distribution of all array probes irrespective of their expression levels. The size of the circles reflects the number of probes.(391 KB PDF)Click here for additional data file.Figure S3Shown are the original signal intensity distributions of exonic and intergenic probes expressed in human testis (A), and the signal intensity distributions of the probe subsets (B). The subsets of probes obtained using this procedure included on average 90% of exonic and 46% of intergenic probes from the original distributions.(87 KB PDF)Click here for additional data file.Figure S4The overlap between tissues is shown for expressed exonic (left) and intergenic (right) probes. The bars represent the proportion of overlap between expressed probes calculated in all pairwise comparisons among the four tissues for probes located on the positive (A) and on the negative (B) chromosome strands. The red colored areas inside the bars show the overlap expected by chance.(149 KB PDF)Click here for additional data file.Figure S5The distances were calculated either including (A) or excluding (B) probes mapped to putative genes. The distance distributions are shown separately for brain (upper left panel), lymphoblastoid cell lines (lower left panel), heart (upper right panel), and testis (lower right panel). For all tissues the distance range presented on the figure includes more than 90% of all distances. Red denotes the distance distribution expected by chance.(486 KB PDF)Click here for additional data file.Figure S6p < 0.05, corrected for multiple testing). The mean of the bars shows the mean correlation coefficient, while the bar borders represent a 75% confidence interval. The error bars depict a 95% confidence interval of the correlation coefficient calculated by bootstrapping the list of intergenic probes within each region 500 times.Correlations are shown for intergenic (left four bars) or exonic probes (right four bars). The letters and colors indicate tissues . The width of the bars is proportional to the number of the ENCODE regions showing significant correlations Click here for additional data file.Figure S7Shown are expression differences between species in seven genomic regions measured on tiling arrays (red) and by Q-PCR using as a template cDNA generated using random primers (dark blue) or poly(dT) primer (light blue). Tested regions one and seven correspond to intergenic transcripts; the remainder, to the transcripts of known genes. The error bars represent the standard error of the mean.(112 KB PDF)Click here for additional data file.Figure S8Shown are the average DNA sequence conservation scores  calculated for intergenic probes expressed in all four tissues (A), in at least one tissue (B), not expressed in any of the four tissues (C), and for exonic probes (D). The error bars depict a 95% confidence interval of the mean conservation score, calculated by bootstrapping the sequence conservation scores within each list 1,000 times.(74 KB PDF)Click here for additional data file.Figure S9Shown are the average expression divergence (A and B) and divergence-to-diversity ratio (C and D) between humans and chimpanzees in brain (blue), heart (black), and testis (red) for exonic (A and C) and intergenic (B and D) probes measured in a sliding window within a signal intensity range from 50 to 1200.(323 KB PDF)Click here for additional data file.Figure S10Shown are average expression divergence (A) and divergence-to-diversity ratio (B) between humans and chimpanzees in brain (yellow), heart (blue), and testis (red) for exonic and intergenic probes with a signal intensity lower than 50 in both species. The colored areas indicate 95% confidence intervals based on bootstrapping 1,000 subsets of exonic and intergenic probes. The darker shades indicate expression on the positive DNA strand, while the lighter shades indicate expression on the negative DNA strand. The symbols represent the mean value for the tissue on either the positive (\u0394) or the negative (\u25cb) strand.(237 KB PDF)Click here for additional data file.Table S1(18 KB XLS)Click here for additional data file.Table S2(19 KB XLS)Click here for additional data file.Table S3(29 KB XLS)Click here for additional data file.Table S4(19 KB XLS)Click here for additional data file.Table S5t-test p < 0.001, FDR < 0.05)(Student's (357 KB XLS)Click here for additional data file.Table S6(21 KB XLS)Click here for additional data file.http://www.ebi.ac.uk/arrayexpress) under the accession number E-TABM-136.All primary expression data are publicly available at ArrayExpress ("}
+{"text": "Microorganisms display vast diversity, and each one has its own set of genes, cell components and metabolic reactions. To assess their huge unexploited metabolic potential in different ecosystems, we need high throughput tools, such as functional microarrays, that allow the simultaneous analysis of thousands of genes. However, most classical functional microarrays use specific probes that monitor only known sequences, and so fail to cover the full microbial gene diversity present in complex environments. We have thus developed an algorithm, implemented in the user-friendly program Metabolic Design, to design efficient explorative probes.Sphingomonas paucimobilis sp. EPA505 using a designed microarray of 8,048 probes. As expected, microarray assays identified the targeted set of genes induced during biodegradation kinetics experiments with various pollutants. We have then confirmed the identity of these new genes by sequencing, and corroborated the quantitative discrimination of our microarray by quantitative real-time PCR. Finally, we have assessed metabolic capacities of microbial communities in soil contaminated with aromatic hydrocarbons. Results show that our probe design  can be used to study a complex environment efficiently.First we have validated our approach by studying eight enzymes involved in the degradation of polycyclic aromatic hydrocarbons from the model strain We successfully use our microarray to detect gene expression encoding enzymes involved in polycyclic aromatic hydrocarbon degradation for the model strain. In addition, DNA microarray experiments performed on soil polluted by organic pollutants without prior sequence assumptions demonstrate high specificity and sensitivity for gene detection. Metabolic Design is thus a powerful, efficient tool that can be used to design explorative probes and monitor metabolic pathways in complex environments, and it may also be used to study any group of genes. The Metabolic Design software is freely available from the authors and can be downloaded and modified under general public license. The second encodes a polypeptide 90% similar to a putative ferredoxin component of dioxygenase, named BphA3 [EMBL: BAC65428], involved in various steps of the process of PAH degradation for the electron transfer from reductase to dioxygenase complex [BAC65427] and AhdA1c [EMBL: BAC65426], two components of a terminal oxygenase involved in the monooxygenation of salicylate, a metabolic intermediate of PHE, to catechol [CAG17576 and CAG17577] of the ring-hydroxylating dioxygenase (phnA1a and phn2a respectively) of Sphingomonas sp. CHY-1, involved in the conversion of several PAHs into their corresponding dihydrodiols [BAC65450] of Sphingobium sp. P2 and involved in the electron transfer in association with BphA3 [FN552592] encodes a partial 107 amino acid sequence 97% similar to a 1,2-dihydrodiol-1,2-dihydroxy-dehydrogenase named BphB [EMBL: ABM79802] of Sphingobium yanoikuyae B1.  7.83) compared with the others targeting the same region. We hypothesize that the strongest SNR' probe perfectly matched, or is the closest sequence to targeted genes. Using sequences of  complex . Interes complex , but a ccatechol . Two gendrodiols . The thith BphA3 . The lasbphA3 and ahdA2c genes probe designs allow the specific identification of these genes. For the genes phnA1a, phnA2a, ahdA1c, bphB and bphC, only one region can be considered specific for the identification of the genes. Finally, for ahdA4 gene, as no probes give positive signals, we can then hypothesize that ahdA4 is not expressed or is weakly expressed (under the detection threshold) in our culture conditions. We can also postulate that absence of signal might reflect a low sensitivity of these selected probes targeting ahdA4.Comparison of these gene sequences with the microarray probes shows that our design strategy is efficient to detect, with no prior sequence assumptions, targeted genes from complete metabolic pathways. As expected, for each gene, different probes give positive signals in agreement with the gene sequence composition. Furthermore, among the thirteen probes (targeting both regions of the eight genes) giving the highest signals, nine probes perfectly match strain EPA505 targeted gene regions Table . Thus thTo conclude, these results confirm that our design strategy is useful and efficient for the targeted genes studied. These data also show that it is essential to select at least two specific regions for each studied gene that should be experimentally validated to ensure accurate identification. Nevertheless, a majority of selected regions is useful for the design of efficient probes that perfectly hybridize with their targets and show the strongest signal on the microarray.As described previously, the applied design strategy lets us to detect targeted genes from the studied metabolic pathway without prior assumptions. It is thus of interest to test whether our DNA microarray is able to evaluate mRNA levels semi-quantitatively during biodegradation kinetics with PHE, FLA and a mixture of the two pollutants as sole carbon and energy source. A control experiment with glucose as sole carbon and energy source is also conducted. For these four conditions, total RNAs are extracted from pure cultures of strain EPA505 at different times of the kinetics . According to the explorative probe validation conclusions (see previous section), only the most efficient probes targeting each of the eight genes in response to pollutant exposure are considered. In addition, to evaluate the gene expression level, a quantitative reverse transcription PCR approach is also developed for the selected genes during the same times of the kinetics.ahdA1c, bphC and bphB probes, positive SNR' values are also obtained with specific probes after 3 h of growth. After 6 h of culture with FLA, no positive probe signal (SNR' > 3) is detected, as in glucose-growth conditions  for the different times of the kinetics studied as shown in Additional file  \u00b1ahdA4 gene, under the SNR' threshold and for bphC and bphB, just above the SNR' threshold, after 3 h of culture  with FLA, but not after 3 h of culture. Finally, with a mixture of the two pollutants, high positive signals are detected, except for the  \u00b1  0.40).phnA1a, phnA2a, ahdA1c, ahdA2c, bphB, bphC and bphA3 with DNA microarray and quantitative reverse transcription PCR approaches, demonstrating the efficiency of probes designed using Metabolic Design software. Thus DNA microarrays using Metabolic Design can be used to perform semi-quantitative monitoring of gene expression.At the same time, a quantitative reverse transcription PCR based approach is used to precisely describe the gene expression during the growth kinetics. Results show the same expression profiles as those observed with DNA microarray experiments  (data not shown). We can then hypothesize that other positive probe sequences presenting a slight homology with available phnA2a sequences might have targeted phnA2a unknown genes, consistent with the explorative purpose of these probes.Among the 8,048 designed probes targeting the eight genes, 358 give positive signals (SNR' > 3) after hybridization with total DNA  among 204 other positive probes for this gene. As for  \u00b1  0.34). The same positive results are obtained with probes specific to strain EPA505 genes: 3.12  \u00b1phnA2a, 4.07   1.00 for  \u00b1ahdA2c, 4.33   0.27 for  \u00b1bphC and 7.06   1.14 for  \u00b1bphA3, suggesting the presence of bacteria closely related to strain EPA505.  1.22 for phnA1a gene in the polluted soil. We choose to amplify, with a PCR approach, phnA1a genes using degenerate primers (data not shown). The PCR products are then cloned, and eight clones are sequenced. Among these eight sequences, seven showing high similarities with phnA1a genes are then compared with our probe sequences. This comparison reveals multiple mismatches (data not shown), impeding hybridizations with our probes. This result indicates a marked divergence of this gene family. Our first design focused on phnA1a genes related to Sphingomonas. For a broader discovery of gene diversity, we will need to design probes that take into account more exhaustively the most complete sequence diversity in databases .Surprisingly, no probe can detect We have developed and validated a new algorithm named Metabolic Design. This software can be used to design efficient explorative probes for functional DNA microarrays. Previously to probe design, users have to extract from public (Swiss-Prot and TrEMBL) or personal databases, protein sequences of interest. Results are then integrated in a user-friendly, intuitive interface. All databases used for the application can be selected by the users and they can also integrate personal data. Such flexibility is generally not available, for example with current metabolic reconstruction tools, such as the 'Pathway Tools Software', initially developed for the EcoCyc project , or KEGGIn order to bypass the faulty annotations found in automatically filled databases, and to allow the exhaustive exploitation of all the currently available protein sequences, the mining step is performed using similarity search. However, such approach presents another major drawback. Indeed, in some cases, not all proteins with a similar function have similar primary structures. Thus a future development of Metabolic Design will be the replacement of the BLASTp step by a Pattern Hit Initiated BLAST (or PHI-BLAST) step coupled with PRODOM data  . PHI-BLAin silico. Specific probes deduced from defined degenerate probes thus allow the targeting not only of known gene sequences but also of new ones that encode the same protein sequences. These explorative features are not offered by other tools such as OligoArray 2.0, YODA or HPD  , [EMBL: FM882254] (encompassing phnA1a and phnA2a gene sequences), [EMBL: FM882253] (encompassing ahdA4 gene sequences) and [EMBL: FN552592] (encompassing bphB gene sequences).The nucleotide sequences reported in this study have been deposited in the database under accession numbers: [EMBL: GUI: graphical user interface; IUPAC: international union of pure and applied chemistry; PAH: polycyclic aromatic hydrocarbon; PHE: phenanthrene; FLA: fluoranthene; SNR': signal to noise ratio; ORF: open reading frame; CDS: coding DNA sequence; RT-PCR: reverse transcription- polymerase chain reaction; FTP: file transfer protocol; JRE: java runtime environment; SQL: structured query language; UTR: untranslated region, aRNA: antisense RNA; DMSO: dimethyl sulfoxide; cDNA: complementary DNA.Project name: Metabolic DesignProject homepage: ftp://195.221.123.90/Operating system: Windows (32-bit) onlyProgramming language: Java and PerlOthers: The Java runtime environment (JRE) Version 1.4 or higher, Perl Version 1.5 or higher and an SQL database such as Oracle 9i must be installed.License: Free for non-commercial use. Source code available upon request.in silico analysis. ST, EP, OG and PP planned the study and wrote the manuscript. JT, EP, OG, DB, CBP and PP have given final approval of the version to be published. All authors read and approved the final manuscript.ST, EP, AM and OG carried out the experimentations and have participated to analysis and interpretation of data. FG, EP, ST and OG have made script developments, updates and optimizations of Metabolic Design. ST, EDB, FG, EP and OG performed SNR' profiles detected with microarray experiments and transcript numbers profiles detected with quantitative RT-PCR assays. SNR' profiles detected with microarray experiments (LEFT), and transcript copy number detected per ng of total RNA with quantitative RT-PCR assays (RIGHT) for eight genes: (A) phnA1a; (B) phnA2a; (C) ahdA1c; (D) ahdA2c; (E) bphB; (F) bphC; (G) bphA3; and ahdA4 (H) during PAH biodegradation at different times with strain EPA505. PHE: grey squares, FLA: triangles, PHE + FLA: circles, glucose: open diamond. Error bars indicate the standard deviation of measures.Click here for filePAH composition detected in the contaminated soil S3. These data are proprietary data given by BioBasic Environnement and give the quantity of detected PAHs in mg/kg of dry soil in the contaminated soil studied.Click here for fileBackground noise calculation description. Background noise is determined according to 'RANDOM probes response' of Nimblegen microarrays. Our method takes into account the background noise which is characterized by two components: its position and its dispersion.Click here for fileIdentification of four catabolic genes clusters from the model strain EPA505. Physical maps of four clusters  of catabolic genes involved in PAHs biodegradation from strain EPA505. Size of genes and intergenic spaces is indicated as well as position of primers used for PCR amplifications.Click here for filePrimer sets used for detecting catabolic genes involved in PAHs degradation and to generate the gene DNA matrix. The DNA matrix is used to build the standard curve for quantitative real-time PCR assays in strain EPA505. *: xylX and nahD are used to characterize complete sequences of bphC and ahdA1c. Nomenclature: M: A and C; R: A and G; W: A and T; S: G and C; Y: C and T; K: G and T; V: A, G and C; H: A, C and T; D: A, G and T; B: G, T and C; I: A, C, G and T.Click here for filePrimers used for reverse transcription and quantitative real-time PCR assays. List of primers used for reverse transcription and subsequent quantitative real-time PCR assays. Amplification sizes are also given for each targeted gene.Click here for file"}
+{"text": "DNA microarray technology allows the analysis of genome structure and dynamics at genome-wide scale. Expression microarrays (EMA) contain probes for annotated open reading frames (ORF) and are widely used for the analysis of differential gene expression. By contrast, tiling microarrays (TMA) have a much higher probe density and provide unbiased genome-wide coverage. The purpose of this study was to develop a protocol to exploit the high resolution of TMAs for quantitative measurement of DNA strand-specific differential expression of annotated and non-annotated transcripts.S. pombe 1.0FR tiling microarrays to generate custom Chip Description Files (CDF) in order to compare their efficiency. We experimentally tested the potential of our approach by measuring the differential expression of 4904 genes in the yeast Schizosaccharomyces pombe growing under conditions of oxidative stress. The results showed a Pearson correlation coefficient of 0.943 between both platforms, indicating that TMAs are as reliable as EMAs for quantitative expression analysis. A significant advantage of TMAs over EMAs is the possibility of detecting non-annotated transcripts generated only under specific physiological conditions. To take full advantage of this property, we have used a target-labelling protocol that preserves the original polarity of the transcripts and, therefore, allows the strand-specific differential expression of non-annotated transcripts to be determined. By using a segmentation algorithm prior to generating the corresponding custom CDFs, we identified and quantitatively measured the expression of 510 transcripts longer than 180 nucleotides and not overlapping previously annotated ORFs that were differentially expressed at least 2-fold under oxidative stress.We extensively filtered probes present in Affymetrix Genechip Yeast Genome 2.0 expression and GeneChip We show that the information derived from TMA hybridization can be processed simultaneously for high-resolution qualitative and quantitative analysis of the differential expression of well-characterized genes and of previously non-annotated and antisense transcripts. The consistency of the performance of TMA, their genome-wide coverage and adaptability to updated genome annotations, and the possibility of measuring strand-specific differential expression makes them a tool of choice for the analysis of gene expression in any organism for which TMA platforms are available. The introduction of gene expression DNA microarrays (EMAs) about 15 years ago opened a whole new range of possibilities for studying genome dynamics by making possible the simultaneous analysis of the transcription of all the genes in a genome ).Bacillus subtilis [Saccharomyces cerevisiae [Schizosaccharomyces pombe [Caenorhabditis elegans [Drosophila [Arabidopsis [The generation of high-resolution transcription maps by hybridizing total RNA to TMAs has uncovered the existence of a large variety of RNAs, many of which are non-coding, in a range of organisms that include subtilis , Saccharrevisiae , Schizoses pombe , Caenorh elegans , Drosophosophila , human [osophila  and Arabbidopsis . This unSchizosaccharomyces pombe to measure differential gene expression under conditions of oxidative stress. We also compared our results with those from a previous study using custom-made microarrays based on PCR amplified probes representing over 4500 S. pombe genes [Here we report a probe-filtering protocol to generate custom Chip Description Files (CDF) to process the hybridization signals of TMAs from each DNA strand in a quantitative manner to measure differential transcriptional expression. CDFs can be generated from any genome annotation or any set of probes in a microarray and they allow direct use with the same tools as those used for the analysis of differential expression with EMAs. We experimentally compared the performance of the Affymetrix TMA and EMA platforms hybridized with identical RNA samples from the yeast be genes ,11. Our S. pombe wild-type strain 972 h- were grown under identical conditions to those described by Chen et al. [595 = 0.2 (4 * 106 cells/ml). Two separate cultures developed from independent single colonies were processed in parallel throughout the entire experiment . Cells from a 30 ml volume were collected by centrifugation at 2000 rpm for 2 minutes and the pellet was immediately frozen in liquid nitrogen. Hydrogen peroxide  was added to the rest of the culture at a final concentration of 0.5 mM and incubation was allowed to proceed for 30 minutes, after which 30 ml of culture were processed as above.Cultures of n et al.  in 100 m\u03bcl extraction buffer , 20 \u03bcl phenol/chloroform, 2 \u03bcl 10% SDS, 200 \u03bcl glass beads . Cells were mechanically disrupted in a Fast-Prep device (Savant BIO 101) and the cell lysate was extracted with phenol, phenol/chloroform and chloroform/isoamyl alcohol before precipitation with 0.3 M sodium acetate and ethanol. RNA was resuspended in 50 \u03bcl of sterile water with diethyl pyrocarbonate (SIGMA D-5758) and was further purified with the RNeasy mini kit (Quiagen) following the supplier's specifications.Total RNA was prepared by resuspending the cell pellets in 20 \u03bcg of total RNA was used for cDNA synthesis. Target labelling was performed following the instructions of the Affymetrix GeneChip whole transcript double-stranded target-labelling assay manual. To hybridize the Affymetrix GeneChip S. pombe 1.0FR tiling microarray (TMA), 300 ng of total RNA without rRNA reduction was used for cDNA synthesis. Target labelling preserving the original polarity of RNAs was performed following the instructions of the GeneChip whole transcript sense target labelling assay manual from Affymetrix. Biological duplicates from cells treated and not treated with 0.5 mM hydrogen peroxide were used to hybridize TMAs and EMAs. The Pearson correlation coefficients of the probe hybridization signals between TMA duplicates hybridized with RNA from untreated and hydrogen peroxide-treated samples were 0.997 and 0.996, respectively. In the case of EMAs, the Pearson correlation coefficients were 0.998 and 0.998, indicating minimum variability between duplicates. The complete set of microarray hybridization results is available at the GEO database under accession number GSE19020.To hybridize the Affymetrix Genechip Yeast Genome 2.0 expression microarrays (EMA), 7 For differential expression analyses, microarray probe intensities were processed using the Robust Multiarray Average (RMA) procedure, which includes RMA background adjustment, quantile normalization, and median polish summarization .The segmentation algorithm used to define the boundaries of non-annotated differentially transcribed regions (dTRs) included only probes displaying a difference in the hybridization signal above 0.8 . Probes less than 60 nucleotides apart (approximately 3 tiling probes) were clustered in a single region. Only regions larger than 180 nucleotides with an average hybridization signal difference of all the probes included above 0.8  were selected. Regions meeting these criteria were fused if the distance between them was shorter than 120 nucleotides.Analysis of differential gene expression using microarrays requires the generation of a Chip Description File (CDF), which links each position in the microarray to a specific gene. For the specific analyses addressed in this work, we generated several custom CDFs following the steps indicated in the flowchart shown in Figure S. pombe genome (in which the 1.2 Mb of the rDNA locus was excluded). This step was optimized using a Karp-Rabin algorithm [Saccharomyces cerevisiae genome included in the Affymetrix Genechip Yeast Genome 2.0 expression microarrays. In a third stage, probes mapping to more than one position in the genome are also discarded. In the fourth step, each probe is mapped against the corresponding genome annotation to select only those matching exons or predicted open reading frames (ORF). As a reference, we used the Sanger Centre genome annotation release of July 17, 2009, which includes 5063 genes. The fifth step integrates all remaining probes into probesets that represent the number of ORFs in a given genome annotation. Genes represented by less than four probes in TMAs or EMAs were not incorporated into the final CDFs because this has been reported to be the lowest number of probes able to provide statistically significant results [In the first step, the sequences of probes in the Affymetrix platforms are filtered so that no more that one probe with the same nucleotide sequence will be present in the CDF. In a second step, each single copy probe is mapped against the complete genome sequence used as a reference and those not matching are discarded (this can result from sequence updating after the microarray was designed). This process requires intensive computation since more than a million probes Table  must be lgorithm , adapted results . The CDF results .Following this scheme, we generated three custom CDFs Table :S. pombe 1.0FR tiling array filtered as described above to generate 4972 probesets.1. CDF \"Sp_TMA\". This included probes from the Affymetrix GeneChip 2. CDF \"Sp_EMA\". This included probes from Affymetrix GeneChip Yeast Genome 2.0 filtered to generate 4904 probesets. We used the same genome annotation as in the Sp_TMA CDF to make the results comparable between both platforms.3. CDF \"Sp_PCR_TMA\". To compare results from both Affymetrix platforms and custom designed microarrays developed in the Sanger Centre , as a rehttp://genomics.usal.es/TMADE.The Perl software used and the custom CDFs generated for expression analysis can be downloaded from our web site As shown in Table cdc13 (1449 bp) or taz1 (1992 bp), are represented by 67 and 92 probes, respectively, in Sp_TMA, and by 11 in both cases in Sp_EMA. Only very small genes, such as SPAC11D3.01c (240 bp), would be represented by a lower number of probes in Sp_TMA than in Sp_EMA. This disadvantage would affect 82 annotated ORFs shorter than 240 bp, which represent only 1.6% of all S. pombe genes. Figure The differences in the probe coverage of specific genes in the different platforms are illustrated in Figure S. pombe under conditions of oxidative stress using the TMA and EMA platforms. Figure We undertook a comparative analysis of differential gene expression of Arabidopsis, in which a strong correlation between the performance of EMAs and TMAs for quantitative gene expression was also found [S. pombe genome relative to Arabidopsis or to the fact that repetitive probes were not filtered out in that study.Taken together, these results show that the Sp_TMA and Sp_EMA Affymetrix platforms yielded virtually identical results, thus validating the use of TMAs for the analysis of differential expression of annotated genes. These results are consistent with those reported in a previous study carried out in so found . The facSPBC21C3.19 gene  from each DNA strand Figure . This exe Figure . Two exae Figure  and 5D. e Figure . The quaSaccharomyces cerevisiae have uncovered an unexpectedly large amount of stable and unstable non-coding RNAs, a large fraction of which are transcribed bidirectionally from nucleosome-free regions [S. cerevisiae [The development of DNA microarrays and more recently of deep sequencing technologies has revealed that in addition to protein coding genes, a large fraction of eukaryotic genomes are transcribed. Detailed transcriptome maps in  regions ,18. In orevisiae . The posrevisiae  and is oWe have shown that information derived from TMA hybridization can be simultaneously processed for high-resolution qualitative and quantitative analysis of differentially transcribed regions. The consistency of the performance of TMAs, their genome-wide coverage, and their adaptability to updated genome annotations, together with the possibility of quantitative measurement of the differential expression of non-annotated and antisense transcripts, makes them a tool of choice for the analysis of genome dynamics in any organism for which TMA platforms are available.LQ designed the computational methods and implemented the algorithms. MS performed the biological assays and prepared RNA samples for microarray hybridization. FA designed and supervised the general strategy of the work. LQ and FA wrote the article. All authors analyzed data, discussed results and approved the final version.Schizosaccharomyces pombe growing under oxidative stressDifferential expression of annotated ORFs in . Columns indicate the following: (A) Gene name. Different synonyms for each gene are indicated, (B) number of exons, (C) ORF length, (D) number of probes per probeset in Sp_TMA, (E) Differential expression (log2) in stressed relative to non-stressed cells in Sp_TMA (orange), (F) Corresponding p-value, (G) number of probes per probeset in Sp_EMA, (H) Differential expression (log2) in Sp_EMA (green), (I) corresponding p-value, (J) Differential expression (log2) in Sp_PCR_EMA (blue), (K) number of probes in PCR amplicons used as probes in Sp_PCR_TMA, (L) Differential expression (log2) in Sp_PCR_TMA (purple), (M) corresponding p-value.Click here for fileSchizosaccharomyces pombe under oxidative stressDifferentially transcribed regions (dTRs) in . Columns indicate the following: (A) identification number assigned to each dTR under the experimental conditions used, (B) differential expression (log2) in stressed relative to non-stressed cells, (C) corresponding p-value,  chromosome number, strand polarity and genomic coordinates of dTR initiation and end, (F) dTR length, (G) Empty cells indicate no overlap between dTR and any annotated transcript in the Sanger Centre genome release of July 17, 2009. Different synonyms for each gene are indicated. A symbol < or > preceding the name of the gene indicates that one end of the annotated ORF maps within the dTR and the other lies beyond its starting or end boundary, respectively. Symbols > < flanking the name of a gene indicate that the entire ORF is included within the boundaries of the dTR. Symbols < > flanking the name of the gene indicate that the entire dTR is included within the boundaries of the ORF. The 2328 entries in the table correspond to 2124 dTRs (Column A). The difference is due to the fact that some dTRs overlap with several exons. Out of the 2328 entries, 1546 were differentially expressed at least 2-fold and were distributed as follows: 510 dTRs (33.0%) had an average length of 613 nucleotides and did not overlap with annotated ORFs; 346(22.4%) were entirely included within ORFs and the remaining 690 (44.6%) overlapped partially with annotated ORFs. These data can be accessed from the genome browser on our website http://genomics.usal.es/TMADE/browser.Click here for file"}
+{"text": "Development of high-density microarrays for global analysis of gene expression and transcription factor binding in Streptomyces coelicolor  suggests a novel role for HspR in stress adaptation. in situ-synthesized DNA arrays for the G+C rich bacterium Streptomyces coelicolor. High G+C content DNA probes often perform poorly on arrays, yielding either weak hybridization or non-specific signals. Thus, more than one million 60-mer oligonucleotide probes were experimentally tested for sensitivity and specificity to enable selection of optimal probe sets for the genome microarrays. The heat-shock HspR regulatory system of S. coelicolor, a well-characterized repressor with a small number of known targets, was exploited to test and validate the arrays for use in global chromatin immunoprecipitation-on-chip (ChIP-chip) and gene expression analysis.DNA microarrays are a key resource for global analysis of genome content, gene expression and the distribution of transcription factor binding sites. We describe the development and application of versatile high density ink-jet dnaK, clpB and lon as in vivo targets of HspR, it was revealed, using a novel ChIP-chip data clustering method, that HspR also apparently interacts with ribosomal RNA (rrnD operon) and specific transfer RNA genes (the tRNAGln/tRNAGlu cluster). It is suggested that enhanced synthesis of Glu-tRNAGlu may reflect increased demand for tetrapyrrole biosynthesis following heat-shock. Moreover, it was found that heat-shock-induced genes are significantly enriched for Gln/Glu codons relative to the whole genome, a finding that would be consistent with HspR-mediated control of the tRNA species.In addition to confirming Streptomyces biology.This study suggests that HspR fulfils a broader, unprecedented role in adaptation to stresses than previously recognized - influencing expression of key components of the translational apparatus in addition to molecular chaperone and protease-encoding genes. It is envisaged that these experimentally optimized arrays will provide a key resource for systems level studies of  Streptomyces coelicolor A3(2) ) were designed. For this version of the array all non-coding sequences upstream of protein-encoding genes were selected (sequences where transcription factors are most likely to bind) and multiple 60-mer probes targeting those regions were selected from the 'all possible probes' set. Following this initial selection, a total of 84,268 probes were experimentally tested and the best performing 21,064 probes that represented all upstream intergenic regions (an average of 3 approximately 110 bp spaced probes to each upstream site) in the genome were synthesized on the array. As this array design was developed specifically for ChIP-on-chip experiments, all probe sequences corresponded to one strand only (that in [EMBL:AL645882.2]) since the particular DNA strand was unimportant. (Note that intergenic regions flanked by transcription terminators for convergently transcribed genes were not selected for this array.)All possible 60-mer probes for all targets (both coding and non-coding sequences) in the AL645882.2]). The average spacing of probes in the genome was approximately 135 bp.From the 'all possible probes' set (see above), 964,820 60-mer probes were selected and printed to target all coding and non-coding sequences with minimal distance between the probes and maximal coverage of the genome. Following experimental validation of probe signal and specificity, 43,798 of the best performing probes were selected to give broad coverage. Probes within protein coding sequences corresponded to the mRNA strand for (cDNA-based) detection of gene expression. For intergenic regions the probe sequences corresponded to one strand only  as described in Materials and methods. For these experiments, S. coelicolor was cultivated under non-heat-shock conditions in a rich liquid medium containing 10.3% sucrose to support dispersed growth of the mycelium and provide sufficient biomass for the ChIP protocol; this was to maximize formaldehyde penetration and to determine the genomic distribution of HspR under non-stressed conditions (the 'resting' state). Following the IP reaction, the DNA was recovered, labeled with Cy3-dCTP and then co-hybridized onto the Sco-Chip2-v1 and Sco-Chip2-v2 arrays together with the Cy5-dCTP-labeled total chromatin as reference (Sco-Chip2-v1) or with Cy5-dCTP labeled mock 'no-antibody' IP chromatin (Sco-Chip2-v2) . The results presented in Figures in vivo, to the dnaK, clpB and lon promoter regions and, importantly, have served to identify additional putative HspR targets. The statistical approaches used to score probe enrichment ratios (gene targets) as significant differed between the two array formats because in Sco-Chip2-v1 the probes were focused only on promoter regions while in Sco-Chip2-v2 the probes were relatively evenly spaced across the genome . The targets scored as significant using Sco-Chip2-v1 were dnaK, clpB, lon and SCO5639 and those on Sco-Chip2-v2 were dnaK, clpB, lon and probe sequences between SCO3019-SCO3020 and SCO5549-SCO5550, corresponding, respectively, to the promoter region of the rrnD ribosomal RNA operon and a five-tRNA cluster encoding tRNAGln and tRNAGlu species; if the cut-off threshold was slightly relaxed for the Sco-Chip2-v2 data, then SCO5639 was also identified.For the ChIP-on-chip experiments, samples of 2-v1 because they are non-protein-coding and are positioned between convergently transcribed genes. The discovery that HspR may regulate specific tRNA and rRNA genes is unprecedented and suggests a more global role for HspR in the stress response of Streptomyces. The HspR-specific probe enrichments in the previously known and the new putative HspR targets are shown in Figure The respective nucleotide sequences of the new putative stable RNA targets of HspR had been excluded from Sco-Chip2-v2 design allowed us to detect gene expression using the same probe set. Thus, in Figure hspR disruption mutants relative to the wild-type strain; and the ratio of expression from cultures heat-shocked at 42\u00b0C relative to non-heat-shocked control cultures. It is noted that the observed reduction of relative transcript levels of operonic genes more distal from the operon promoter  threshold of <0.15 ) as a result of heat-shock . The use of such thresholds has been reported elsewhere ).S. coelicolor. The entire set of upstream sequences was then aligned independently to the previously published HspR consensus sequence (5'-TTGAGYN(7)ACTCAA) . The red line indicates the threshold applied to identify enriched probes.Average  plotted as [logClick here for file2 Antibody bound/'No Antibody' bound]. The red line indicates the threshold applied to identify enriched probes.Average  plotted as [logClick here for fileAL645882.2].The anti-HspR-enriched probes are plotted on a linear scale. Open circles indicate the start co-ordinate (relative to genome sequence) of each probe that passed quality control filtering. The genetic organization of each region is indicated below the plot; each arrow represents a coding sequence or stable RNA gene as defined in [EMBL:Click here for fileTabulation of genes significantly up-regulated following heat-shock at 42\u00b0C.Click here for filehspR disruption mutant relative to wild-type.Tabulation of genes significantly up-regulated in an Click here for fileGln/Glu cluster.Partial matches to the HspR-binding consensus sequence within the five tRNAClick here for file2-v2 array.Motif identified from MEME analysis of the HspR targets identified with the Sco-ChipClick here for fileS. coelicolor genome [EMBL: AL645882.2].Over-representation of amino acids in heat-shock induced gene products  relative to all annotated proteins from the Click here for fileSCO3202 (hrdD) and SCO4157 had respective rank products pfp values of 0.12 and 0.13. SCO4410 and SCO5639 were identified as new putative targets for HspR. Average values are plotted from the same two independent biological replicates of each condition used in the array-based expression analysis.Click here for fileAverage values are plotted from the same two independent biological replicates of each condition used in the array-based expression analysis.Click here for fileComparison of expression of heat-shock genes from SMMS surface-grown cultures  in YEME+10.3% sucrose liquid cultures (as used in ChIP-on-chip experiments).Click here for file2-v1 arrays.Probes found to be significantly enriched for HspR on Sco-ChipClick here for file2-v2 arrays.Probes found to be significantly enriched for HspR on Sco-ChipClick here for filerrnD data), with a dotted line along log ratio of 0.An enlarged image of Figure Click here for fileAn enlarged image of Figure Click here for file"}
+{"text": "E. coli through various approaches is above 70, with several hundred more sRNA candidate genes under biological validation. Although the total number of sRNAs in any one species is still unclear, their importance in cellular processes has been established. However, unlike protein genes, no simple feature enables the prediction of the location of the corresponding sequences in genomes. Several approaches, of variable usefulness, to identify genomic sequences encoding sRNA have been described in recent years.Small untranslated RNAs (sRNAs) seem to be far more abundant than previously believed. The number of sRNAs confirmed in in silico comparative genomics and microarray-based transcriptional profiling. This approach to screening identified ~60 intergenic regions conserved between Sinorhizobium meliloti and related members of the alpha-proteobacteria sub-group 2. Of these, 14 appear to correspond to novel non-coding sRNAs and three are putative peptide-coding or 5' UTR RNAs . The expression of each of these new small RNA genes was confirmed by Northern blot hybridization.We used a combination of sra) genes can be found in the intergenic regions of alpha-proteobacteria genomes. Some of these sra genes are only present in S. meliloti, sometimes in genomic islands; homologues of others are present in related genomes including those of the pathogens Brucella and Agrobacterium.Small non coding RNA ( E. coli range from 50 to several hundred  gave no further hits. This first approach confirmed a key point: most sRNAs cannot be detected by inter-phylum primary sequence identity.Most bacterial sRNA searches were conducted using the ed\" form . SurprisE. coli (K12) genome against S. meliloti's chromosome, but only 4.5S and rnpB were predicted by this tool. Finally, we assessed sRNApredict2 . The radiolabelled probes were then purified through MicroSpin G25 columns . The efficiency of probe labelling was monitored in a liquid scintillation analyser (1600 TR Packard).DNase-treated, S. meliloti was grown in LB medium to mid-exponential phase (OD600 nm= 0.8). Total RNA (10 \u03bcg) was extracted, denatured, and subjected to electrophoresis on an 8% acrylamide/bis acrylamide (19:1) denaturing gel (8 M urea) in 0.5 \u00d7 TBE buffer [\u00ae BioDetect\u2122).E buffer . The RNAS. meliloti 1021 genomic DNA, 1.25 units of TaqDNA polymerase (Promega) and 4 pmol of each oligonucleotide in a final volume of 50 ml for 40 cycles . The PCR buffer contained 5 \u03bcl MgCl2 (25 mM), 1 \u00d7 Taq buffer (Promega) and 4.3 \u03bcl dNTPs. The length and quality of each PCR product was assessed by electrophoresis on 2% agarose TBE  gels. The IGRs-PCR probes were dried, resuspended in 30 \u03bcl betaine, 1.5 M-3 \u00d7 SSC and spotted in triplicate onto GAPS II\u2122-coated glass slides (Corning) through robotic arraying .A PCR-based microarray was designed , the \"noise\" value being the average intensity of spots containing spotting buffer. All genes with log2 SNR lower than 2  were considered as untranscribed under the conditions tested and their value changed to 0 (filtering). The resulting expression profiles are illustrated as a heat map.The labelled cDNA was resuspended in hybridization buffer (3 \u00d7 SSC/0.1% SDS/50% formamide) and cohybridized with 10 \u03bcg of salmon sperm DNA to the microarray glass slides overnight at 55\u00b0C. The slides were washed for 5 min at 55\u00b0C in wash buffer (2 \u00d7 SSC/0.1% SDS) then 2 min in high-stringency buffer , 2 min in 0.2 \u00d7 SSC (twice) and 2 min in 0.1 \u00d7 SSC, and dried by centrifugation . Hybridized microarray slides were scanned  with independent excitation of the fluorophores Cy5 and Cy3 at 10-\u03bcm resolution. Our microarray time series experiment was planned with a dye-swap design; Dabney & Storey recently showed that a simple average dye-swap removes dye bias without affecting the biological signal and preserves the ordering of true expression means . To deteS. meliloti database used [S. meliloti chromosome, as the reference, and other alpha-proteobacteria genomes: Agrobacterium tumefaciens strain C58 Cereon, Rhizobium etli CFN42, Rhizobium leguminosarum bv. vicae 3841 and Mesorhizobium loti MAFF303099.Intergenic regions (IGRs) were determined as genome areas with no gene annotation on either of the two strands. The ase used  includesase used ) from thase used  was usedS. meliloti chromosome (AL591688) as an input, and applying an IGR length threshold of 7 bp. Parameter 'w' in BLAST was set to 7 in order to refine word detection.ISI  was usedE. coli K12 Refseq file (NC_000913) were extracted, and compared to the chromosomal IGRs of S. meliloti thanks to WU BLAST 2.0, with various e-value thresholds . QRNA was then run with scanning window set to 100 nt.In order to produce the input alignment file for QRNA v.2.0.3.c , the 61 S. meliloti chromosome ORF list (NC_003047.ptt) from the Refseq repository [S. meliloti. The t/rRNA database was compiled from data in the TIGR_CMR RNA list [S. meliloti sequencing consortium website [S. meliloti sRNA training set was generated from data available at the corresponding RFAM database page [Agrobacterium tumefaciens str. C58 and Rhizobium etli CFN 42. Consequently, two blast output files were generated, where S. meliloti intergenic regions were compared to the cited genomes using WU BLAST 2.0 with same parameters as Livny [-5, V = 10000 et B = 10000). Finally, databases of regions of predicted conserved secondary structure were produced using QRNA, again with parameters set to the same values as in [sRNAPredict2  was testpository . The \".cRNA list  and from website . The ter website ,89. The ase page . Two refas Livny   alignment and corresponding synteny results. The data provided shows the results of the search for small regions of homology between alpha-proteobacteria.Click here for fileArtemis Comparison Tool (ACT) screenshots alignment and corresponding synteny results. The data provided presents the results of the ACT comparisons.Click here for filein vivo validation and their features.Intergenic regions included in this study. The data provided lists the intergenic regions selected (sra candidates) for Click here for file2SNR) of IGRs candidates. The data provided compiles the normalized expression levels for the candidates.Normalized expression levels (logClick here for fileTargetRNA results. The data provided shows the results of the target analysis for each valid sra gene.Click here for fileAlifold and RNAz secondary predictions. The data provided presents the alternative structures predicted for sra genes depending on the tools used.Click here for fileS. meliloti and related alpha-proteobacteria. The data provided features the multiple loci of sra41 in subgroup-2 alpha-proteobacteria.sra41 multiple loci in Click here for fileStrains used in this study.The data provided references the different bacteria used in this study.Click here for fileOligonucleotides used in RACE and Northern assays. The data provided presents the oligonucleotide sequences used in RACE and Northern assays.Click here for fileOligonucleotides used in microarray design. The data provided presents the oligonucleotide sequences used in the microarray design.Click here for file"}
+{"text": "Arabidopsis thaliana tiling array, which has 2.85 million probes throughout the genome, to measure genetic differentiation between populations of Arabidopsis lyrata growing on granitic soils and those growing on serpentinic soils. The significant overrepresentation of genes involved in ion transport and other functions provides a starting point for investigating the molecular basis of adaptation to soil ion content, water retention, and other ecologically and economically important variables. One gene in particular, calcium-exchanger 7, appears to be an excellent candidate gene for adaptation to low Ca\u2236Mg ratio in A. lyrata.Serpentine soil, which is naturally high in heavy metal content and has low calcium to magnesium ratios, comprises a difficult environment for most plants. An impressive number of species are endemic to serpentine, and a wide range of non-endemic plant taxa have been shown to be locally adapted to these soils. Locating genomic polymorphisms which are differentiated between serpentine and non-serpentine populations would provide candidate loci for serpentine adaptation. We have used the  CollinsiaCerastiumPinusArabidopsis lyrata, a perennial self incompatible crucifer, grow on serpentine outcroppings, interspersed with populations on other soil types such as granitic outcroppings and sand dunes A. lyrata, its proximity to the genetic model organism A. thaliana provides an opportunity to locate polymorphisms which are associated with the serpentine soil habitat. The role of these polymorphisms in serpentine adaptation, if any, can then be experimentally investigated.Serpentine soil mosaics are a classic context for ecological adaptation A. lyrata to A. thaliana Affymetrix tiling arrays, we can measure genetic differentiation between soil types at 2,853,369 probes throughout the A. lyrata genome. When DNA is hybridized to the array, probes which overlap a polymorphic SNP or indel will hybridize poorly in individuals with the mismatched allele A. thaliana , an induced loss of function mutation in the calcium-proton antiporter cax1 enhances survival on soils with a low Ca\u2236Mg ratio A. lyrata would provide clear candidate loci for adaptation to low Ca\u2236Mg ratio. Second, we can use the natural distribution of genetic variation between environments to investigate the functions of unannotated genes and non-coding features. If an uncharacterized locus consistently assorts with soil ion content, for example, then it can be hypothesized to interact with this environmental variable to determine fitness. Third, we can use genetic differentiation at genes with known function to form hypotheses about other environmental differences which may be important in nature. Although abiotic factors are thought to be the most important drivers of serpentine adaptation, if differentiation is also found in genes coding for anti-microbial compounds, anti-predatory compounds, or proteins which mediate competitive interactions, then parasitism, predation, or competition can be inferred to be important.By hybridizing genomic DNA from A. lyrata to the A. thaliana tiling array. This has allowed us to locate many polymorphisms which are differentiated between soil types, including excellent candidates for adaptation to soil conditions. We do not mean to imply that selection is the only force which leads to differentiation between populations, as stochastic demographic forces may also lead to correlated distributions of genetic polymorphisms and environmental conditions, especially in this small sample of populations In the current work, we have hybridized genomic DNA from two serpentine and two granitic populations of A. lyrata localities, DNA from three individuals from each of the four localities . On the array, each perfect match (PM) probe that matches the genome is adjacent to a mismatch (MM) probe, which has a mismatched base at the middle base pair. Comparison of normalized \u03a3(PMi) and \u03a3(MMi) intensities over the i chips for each probe indicates that the probes matching the draft A lyrata genome are the most sensitive markers of DNA differentiation, as expected (i)\u2212\u03a3(MMi) is positive for 98.8% of probes. For the other 2,481,727 probes on the array, the \u03a3(PMi)\u2212\u03a3(MMi) is positive at 65% of probes. This indicates that probes matching the draft genome are the most sensitive markers for detecting DNA differentiation, but also that the 2.48 million other unique probes contain useful information.To map differentiated polymorphisms between serpentine and granitic calities  was fragexpected . For thet-test p-value for between soil types at each array probe; the distribution of p-values for probes with a perfect match in the draft A. lyrata genome is shown in p-values: 2402 probes have p<0.001, whereas only 371 are expected by chance. For comparison, t-tests were computed between the other two possible combinations of our four collections. Dividing the samples along these additional axes revealed a small excess of probes with low p-values in both cases . When the probes with 0.01<FDR<0.05 are excluded to generate a more conservative list, the number of GO associated genes drops to 263. As noted in the introduction, additional data is required in order to determine how many of these genes are differentiated due to spatially varying selection. We can, however, use the annotations of differentiated loci to prioritize specific genes and molecular functions for further investigation. We find that the genes which are differentiated between soil conditions are associated with a non-random subset of biological functions (p<0.001) and transporters (p<0.001) are very overrepresented among differentiated genes. GO terms which are obvious candidates for adaptation to soil salt and nutrient content include potassium ion transport (p\u200a=\u200a0.006), cellular calcium ion homeostasis (p\u200a=\u200a0.008), and cation transport (p\u200a=\u200a0.03). These genes provide a starting point for understanding the molecular basis of adaptation to known environmental differences, such as low Ca\u2236Mg ratios, and also may indicate additional unappreciated environmental variables. An example of this second category is the differentiated gene AT4G10380, which responds to boron limitation in A. thalianaThe combined list of significantly differentiated loci (probes with FDR<0.05 and windows with FDR<0.001) includes 586 genes, 545 of which are annotated with at least one function in unctions . Membrana priori candidates for soil adaptation: one of the most overrepresented GO terms is microtubule-based movement (p<0.001). Six genes with this function are differentiated, including a putative kinesin heavy chain and four genes which share a kinesin motor protein domain. It is possible that these loci interact with spatially varying environmental variables either directly (gene-by-environment interactions), or to compensate for deleterious side effects of adaptive change at other loci (gene-by-gene-by-environment interactions). An alternative, neutralist, explanation for the overrepresentation of GO terms among differentiated loci is that genes differ in polymorphism rates, and genes with more polymorphism are more likely to have some polymorphisms which are differentiated due to stochastic forces. It should be noted that the overrepresentation of GO terms is not specific to the granitic vs. serpentine population comparison. We also conducted GO tests between the other two combinations of our four populations (serp1+granite1 vs. serp2+granite2 and serp1+granite2 vs. serp2+granite1). In order to have the same power for all tests, the GO test of serpentine vs. granitic soils was re-computed using only the 751 significant probes, and compared to the probes with the lowest 751 t-test p-vales from the other two combinations of the data. For the significant probes, 33 GO terms are significant, while in both other combinations of the data, many different GO terms are significant . Whether this is due to spatially varying selection on different process along different geographic axes, or simply variation in neutral differentiation rates remains to be determined.We also discovered suites of related genes that would not be obvious cax1 in A. thaliana is the only characterized mutation which enhances survival on soils with low Ca\u2236Mg ratios cax7 (AT5G17860), contains a significantly differentiated probe in our analysis. This gene, though functionally distinct from cax1A. lyrata. We sequenced 1.3 Kb of this gene in a larger sample of plants from the four A. lyrata collections and found a small region of high differentiation towards the 5\u2032 end of the coding sequence . Although this gene contained a significant (FDR\u200a=\u200a0.04) probe, this probe did not overlap a differentiated polymorphism and is therefore a false discovery. This gene was in our least significant category, 0.05>FDR>0.01, which is expected to include some false discoveries. Including these probes also increases the sensitivity of the analysis, however: the significant probe in cax7 had a FDR\u200a=\u200a0.011, and would have also been excluded had we only used a threshold FDR of 0.01. Despite reduced power when probes with 0.01<FDR<0.05 are excluded, 20 GO terms remain significant, including cellular calcium ion homeostasis. The conservative and comprehensive lists of significant probes, regions, genes and GO terms are available as supplementary data . The average intensity of the 99 probes in this region in granitic plants is 1.2 , while the serpentine plants have an average intensity of 2.6, which appears to indicate that there is one copy of this region in most granitic individuals and two or three copies in most serpentine individuals. Other cases appear to be differentiated deletions: there are 21 probes which overlap the gene AT5G27100, one of three significantly differentiated ligand-gated ion channels. The average intensity of these probes is 0.986 on serpentine, near the median of all probes, and 0.167 on granite. PCR amplification of this gene from an expanded sample of 48 individuals validated the presence of a deletion with higher frequency in granitic populations: deletion frequencies are 0.79 and 0.65 for the granitic collections and 0.24 and 0.38 for the serpentine collections. The existence of CNVs whose frequencies are associated with environmental conditions, including large duplications of many genes, provides an excellent opportunity to study the influence of selection in the initial stages of gene duplication Drosophila melanogaster also found a considerable contribution of copy number variation to population differentiation, including large variants that affected multiple genes A. halleriMany of our significant regions appear to be large differentiated duplications and deletions . By comparing the intensity of probes in each significant region to an expected distribution generated using probes from all significant regions, we established that 15 of the 71 differentiated regions are likely CNVs , we restricted our analyses to the two patches which we estimate contained over 10,000 individuals. The two non-serpentinic populations sampled are the only ones approximately equidistant to the two serpentinic populations that we could find based on herbaria records  and our own search; the other five sites with herbaria collections on non-serpentinic substrates we visited had disappeared due to ecological succession or human alteration. The serpentinic sites are part of the Nature Conservancy's state line serpentine barren complex in Chester County, Pa, and Soldier's Delight Natural Environment Area, Baltimore County, Md. Granitic sites were located at the Lock 12 recreation area on the Susquehanna river , and Lancaster County Park .Seeds were collected at two serpentine sites and two granitic sites in close proximity . Althouget al. 2008. To fragment DNA, we created a master mix of DNase I (Promega), One-Phor-All buffer (Amersham Biosciences), and Acetylated BSA (Invitrogen); the amounts added per sample were 4 \u00b5l 10X One-Phor-All, 0.14 \u00b5l Acetylated BSA, and approximately 0.085 \u00b5l of DNase per ug of DNA. The precise amount of DNase added per sample depends on the batch of enzyme: our goal was to calibrate the reaction to obtain fragmented DNA of approximately 50 bp, with low variance in fragment size. Fragmentation was performed in a MJ Research PTC-200 thermocycler at 37 C for 16 min, 99 C for 15 min, and 12 C for 15 min. Samples with similar intensities and fragment sizes (approximate fragment size\u200a=\u200a50 bp) were labelled with a master mix of Biotin-N6-ddATP (Enzo) and RTdT enzyme (Promega) to each sample. RTdT was diluted from 30 U/\u00b5l to 15 U/\u00b5l enzyme before use by mixing RTdT enzyme, RTdT 5X buffer, and water at a ratio of 5\u22361\u22364. For each sample to be labelled, 2 \u00b5l of Biotin and 3 \u00b5l of RTdT were added to the master mix. Labelling was accomplished in a thermocycler at 37 C for 90 min, 99 C for 15 min, and 12 C for 5 min. Microarrays were hybridized at the Affymetrix core facility in the UC Davis Genome Center using standard conditions for this array.DNA was extracted from three plants from each of the four populations using Qiagen plant mini-kits and amplified using rolling-circle amplification with the Qiagen Repli-G kit. After phenol-chloroform clean up and ethanol precipitation, 10 \u00b5g of DNA from each sample was fragmented with DNase following Turner A. thaliana reference genome and retained these probes for analysis . We spatially normalized array intensities at these probes by dividing each array into 1600 subarrays of 64 by 64 probes, and divided the log(intensity) of each probe by the median log(intensity) of the local 64\u00d764 probe window. We further normalized these values using quantile normalization in R A. lyrata genome .Using NCBI megablast, we determined that 2,853,369 array probes have a single perfect match in the TAIR7 t-tests were computed for each probe. We first analyzed only the probes with a match to A. lyrata, and corrected these p-values for 371,642 multiple tests. We used three significance thresholds in order to have inclusive and conservative lists of differentiated probes: a bonferroni-corrected p<0.05, and two false discovery rate (FDR) thresholds: FDR<0.01 and FDR<0.05 t-test p-values for the remaining 2,481,727 probes, correcting these values for the greater number of multiple tests, and discovered additional probes significant at the three thresholds. We combined these two sets of significant probes to generate a list of differentiated loci at the three levels of stringency, which are available in p-value * number of multiple tests / p-value rank), and are therefore estimates. These estimates are based on technical, and not biological, false discovery: a probe with low specificity may truly detect differentiation, but not at the expected location.To find differentiated polymorphisms, A. lyrata genome. There were 5,338 probes with FDR<0.30 (p<0.003): these probes have high individual rates of false discovery, but can be used as markers to find regions of the genome that are enriched for differentiated probes. Using permuted chromosomes to generate a null distribution, we mapped windows of 5, 10, and 20 probes that are significantly enriched for candidate markers. Each window recorded the average probe FDR, and compared this value to the permuted chromosomes. Probes with FDR>0.30 were considered to have an FDR\u200a=\u200a1.00, as this was previously found to make the analysis more conservative To increase our power to find differentiated loci, we used a sliding window analysis to determine if probes with low FDR are clustered in the genome. For this analysis, we only used probes with a single perfect match in the draft A. lyrata reference genome, we determined which genes overlapped our differentiated probes and windows; a gene was considered to overlap a differentiated locus if any portion of its transcript overlaps a significant probe or window. Significance of gene ontology (GO) terms among our significant genes was determined in two complementary ways: a binomial sampling test, and a permutation test . First, we compared the observed proportion of significant genes in each GO term to an expected number, which is the binomial sampling probability of sampling an equal or larger number of genes in each category given the number of significant genes in the genome. For the permutation test, we simulated our discovery procedure by randomly sampling probes and genomic regions of the same size and number as our significant probes and regions. For example, in the primary analysis presented below, there are 751 significant probes and 71 significant regions. To generate a null distribution of genes for this analysis, we sampled this same number of probes and regions , and determined which genes overlap the random sample. We considered a GO term to be significantly overrepresented if the observed sample has more genes in the given term than 5% of 500 simulated data sets, and less than 5% probability of occurring by chance in the binomial sampling test.Using the TAIR7 annotation of the p<0.001). This permutation controls for many possible confounding effects. For example, if all derived SNPs were found on serpentine, then granitic populations would have higher average intensities in significant regions; because our permutation samples only from significant regions, the candidate CNV are significantly beyond any such effect.If serpentine and granitic populations are equidistant to the reference genome, then any given differentiated probe should have higher intensity on serpentine half the time, and higher intensity on granite half the time. In some significant regions, however, all probes are more intense on one soil type, consistent with differentiated copy number variants (CNVs). Differentiated regions are considered candidate CNVs if the ratio of mean hybridization intensity for that region is more extreme than expected. We extracted the raw  intensity values for all probes in each significant region, and computed a mean intensity for serpentine and granitic populations. These means were minimally normalized by dividing by the median intensity for all probes for each soil type, and the ratio of the normalized means was used as the test metric. An expected distribution of ratios was created by randomly sampling the number of probes in the test region from the set of probes from all significant regions. Regions were considered candidate CNVs if their ratio was more extreme than 9,990/10,000 random sets , and two were directly sequenced. Population genetic analysis of sequenced loci was done in DNAsp Data Set S1Complete lists of differentiated probes, regions, genes, and GO terms at multiple significance threshold. (0.03 MB PDF)Click here for additional data file."}
+{"text": "SOX2 (SRY [sex determining region Y]) box 2) and OTX2 (orthodenticle homeobox 2), in individuals with developmental eye disease. We conclude that MLGA has the potential to be a useful technique in diagnostic research for the identification of deletions or duplications of known genes due to its speed and relatively low cost.Whole gene deletions or duplications are an important cause of genetic disease and phenotypic variation. Targeted techniques for the routine testing of gross rearrangements have become essential tools for diagnostic researchers with the search for the most cost-effective and efficient tool assuming high priority. We used the new selector technique, MLGA (multiplex ligation-dependent genome amplification), to confirm deletions in two genes,  SOX2 (SRY (sex determining region Y)-box 2), OTX2 (orthodenticle homeobox 2) and BMP4 (bone morphogenetic protein 4), genes involved in severe eye abnormalities  and SRY [sex determining region Y], respectively) to allow for experimental validation in the form of sex confirmation. The five positive cases with known deletions , SOX2OT (SOX2 overlapping transcript) (GenBank RNA accession number BC041898.2), and autosomal control gene, NPM1  (GenBank accession number NM_002520.5). Second, newly designed selector probe and fragment sequences were uploaded to the BLAT alignment tool [SOX2 (SOX2OT) was selected for interrogation due to the relative ease of optimization compared with the GC-rich SOX2 itself. In addition to the control probe, NPM1, mentioned above, selector probes for control genes, MADH4 (mothers against decapentaplegic homolog 4), SRY, and AR, were used as designed by Isaksson et al. [Selector probe design was performed as described by Isaksson et al.  with two browser : OTX2  with previously identified OTX2 deletions and a proportional reduction in the peak area of the SOX2 locus fragment in individuals (cases 4 and 5) with previously identified deletions of the entire SOX2 locus (OTX2 and SOX2 locus deletions). Each result was confirmed in a second assay.The ABI GeneMapper\u00ae traces demonstrated a proportional reduction in the peak area of the OTX2 and SOX2 deletions in developmental eye anomalies. For MLGA to become widely used in a diagnostic service setting, further detailed validation work is required to prove reproducibility in a large number of positive cases and controls. However, the low running costs along with the ease and speed of the assay would be particularly attractive to human genetic diagnostic laboratories requiring regular CNV screens of known disease loci. By amplifying genomic DNA rather than probe molecules, MLGA assays offer the potential to screen at least 50 loci in one assay by using several vectors and multiple labels on the universal primers. Although the routinely used technique, MLPA, is able to analyze this number of loci, MLGA has the potential to do this faster and more economically. Furthermore, MLGA provides great flexibility in the choice of target loci through the library of restriction enzymes available to create fragments for subsequent circularization and amplification.Our results demonstrate that MLGA can identify whole gene deletions in a human diagnostic research setting as exemplified by the diagnosis of human MLGA still requires further validation when multiplexing up to 40-50 loci for it to be deemed as useful as other multiplex targeted approaches (including MLPA) in CNV detection. Moreover, significantly multiplexed assays may require more stringent selector probe design criteria and a more thorough initial optimization of reaction conditions than MLPA since genomic DNA rather than designed probe sequences are amplified. Aside from repetitive elements, most of the genome is accessible to MLGA with only high GC content (>60%) known to affect probe success rates .One significant advantage that MLGA does possess but was not noted in the initial report is the capacity to provide data on insertion-deletion polymorphisms or mutations with a target sequence of interest. For example, a two base pair deletion within a target sequence will give a shifted peak on the GeneMapper\u00ae trace and thus may indicate an avenue for further investigation, such as sequencing. This potential may prove valuable to a researcher screening for mutations as well as deletions, providing a useful \u2018pre-screen\u2019 to detect intragenic deletions or insertions."}
+{"text": "Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples.Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known or novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from  Rapid detection and characterization of bacterial and viral pathogens has become a vital component of our national biodefense strategy. Various detection technologies based on nucleic acid signatures have emerged in the past few years, including TaqMan and Luminex bead based systems. While these technologies are able to rapidly identify selected pathogens at the species or strain level, they do not have the capability to provide broad functional information about known or novel organisms. Characterization of emerging, engineered, or unknown pathogens requires a platform that can assess the virulence and antibiotic resistance mechanisms present in these organisms. One recent approach that has been used successfully to measure other types of microbial capabilities is known as a functional gene array (FGA).A functional gene array is a DNA microarray containing probes targeting sequences unique to genes within families of interest. Family-specific probes are designed to match regions that are conserved among genes in the family, in order to increase the chance of detecting previously unidentified homologs. Small-scale FGAs have been used successfully to measure the presence and activity of key enzymes in environmental samples E. coli strains The FGAs developed to date have focused on specific sets of gene functions, thereby limiting their use to narrowly defined applications. Because of the broad diversity of pathogens and the large number of gene families involved, constructing a functional gene array to detect genes associated with virulence and antibiotic resistance is a much greater challenge. We define virulence-related genes as those whose products affect the ability of a pathogen to infect or survive in the host, are required for expression of other virulence factors, or cause the host direct harm (such as toxins). A high-density oligonucleotide microarray is the only platform available at present that supports simultaneous interrogation of such a wide variety of genes. The approach of using presence or absence of virulence genes as a forensic classifier has been demonstrated in a recent study that used PCR to differentiate several Our laboratory has a NimbleGen array synthesizer that is capable of making arrays with up to 388,000 probes per array, with variable probe lengths ranging from 23 to 85 nucleotides (nt) The other major challenge in constructing functional arrays for detecting virulence genes is the exhaustive computation required to design sensitive and specific probes for hundreds of thousands of gene target sequences. Millions of sequence comparisons are required to find the most conserved regions within gene families and subfamilies, to ensure that probes are selected to span diverse gene sequences that encode similar functions. Thermodynamic binding energy predictions, conservation and uniqueness scores must be computed for millions of candidate probes, in order to select an optimal combination of probes for each target gene family, balancing sensitivity, specificity, and breadth of coverage. The computation of each of these factors is CPU-intensive, requiring that we develop highly efficient algorithms and implement them using high performance computers (HPC) at Lawrence Livermore National Laboratory (LLNL). Our access to HPC facilities has played a crucial role in making high-quality probe design and selection feasible at this scale.In this report, we describe the process used to design our first generation functional array for highly sensitive detection of virulence and antibiotic resistance gene families. We discuss the probe design algorithms, including virulence gene sequence selection, and our protocols for sample preparation, amplification, labeling, hybridization, and data analysis. We present the results from experiments designed to assess whether the array can detect virulence gene orthologs from organisms without perfect match probes on the array, using both targeted mismatch probes and hybridizations to DNA from other organisms. Also, we report the results from limit of detection studies, using known amounts of bacterial DNA spiked into aerosol samples to measure the minimal concentration required for detection of virulence elements against a complex background.E. coli K12 that are avirulent to humans.Gene target sequences were selected from the genomes of the four bacterial strains shown in et al. We selected target sequences from the four genomes by searching for virulence-related proteins using 712 sets of profile hidden Markov models (HMMs). HMM sets were designed or selected by Swan http://www.llnl.gov/pao/news/news_releases/2007/NR-07-04-05.html). For all the predicted gene sequences, existing gene annotations were downloaded from GenBank and correlated with the coordinates of the HMM matches. HMM hits for the same gene family in multiple strains of the same organism had very similar if not identical sequences for all the cases examined.We found matches for 299 of the HMM sets in the four genomes, corresponding to 160 protein families. Some families were represented by multiple paralogs in a given genome, and others by none; in addition, HMM sets within a family sometimes matched distinct but overlapping target sequences, yielding a total of 1,245 matching sequences. The search was performed using the \u201cestwisedb\u201d algorithm of the Wise 2.0 software After selecting and extracting target gene sequences, we designed probes as diagrammed in mT derived by Primer3 from the length, percent GC content and salt concentration. The parameters used for Primer3 are shown in In the first step of probe design, we generated candidate probes from all four organisms for the target gene sequences in a given family using MIT's Primer3 software mT and the minimum free energies of probe-target hybridization (\u0394complementG), probe\u2013probe hybridization (\u0394homodimerG), and probe\u2013self hybridization (\u0394hairpinG). While Unafold is a highly accurate program for \u0394G and mT prediction, it was too slow given our need to calculate mT's and \u0394G's for millions of candidate probes. We created an accelerated version of Unafold that ran more than ten times faster by using more efficient data structures and caching thermodynamic parameter tables in memory rather than reloading them for each probe. We then used the predicted \u0394G's to compute an aggregate \u201c\u0394adjustedG\u201d (described below) for each candidate probe. Candidate probes with unsuitable \u0394G's or Tm's were excluded, unless fewer than 15 candidate probes per target sequence passed all the thermodynamic criteria described above; in this case, candidate probes that failed the filters were included to ensure at least 15 candidates per target.We next used a modified version of Unafold After this initial screening step, we used a custom Perl program to cluster target sequences within each gene family into overlapping \u201cequivalence groups\u201d. Equivalence groups are sets of targets sharing one or more common sequence regions from which probes may be drawn. We implemented a greedy algorithm to select probes from the minimal set of equivalence groups that covered all target sequences in a gene family. The algorithm searches all equivalence groups for a family, finding in each iteration the group containing the largest number of target sequences not already represented by a sufficient number of probes. Probes are selected from the shared sequences in this equivalence group, and the search is repeated until all targets are covered by a minimum number of 15 probes. By prioritizing equivalence groups in this way, the selection process favors probes for regions that are more highly conserved in a gene family.adjustedG and greater dispersal across the target gene sequence.When an equivalence group contained more than 30 candidate probes, we used a custom Python downselection program to choose an optimal subset. The downselection program uses an iterative ranking algorithm favoring probes having lower (more negative) \u0394This process of generating candidate probes, clustering targets into equivalence groups, choosing a minimal set of equivalence groups, and downselecting probes within equivalence groups, was repeated for the target sequences from each gene family.mT between 71\u201391\u00b0C.In addition, we included 2,000 positive control probes from the four genomes. These were designed to be distributed widely across each genome, and to range in length between 50\u201366 nt, in GC content between 40\u201360%, and in Detection of pathogens or gene families represented in a genomic DNA sample requires different probe design criteria than those used for gene expression, ChIP-chip or resequencing purposes. Each target type requires an appropriate balance between probe sensitivity and specificity. Ideally, all probes should be sensitive enough to detect DNA at single-copy concentrations. However, probes intended to distinguish organisms at the species or strain level must be designed to avoid cross-hybridization; while probes used to detect the presence of gene families should allow some degree of mismatch between the probe and target sequence, congruent with the range of sequence variation among orthologs within the family.mT from 66\u00b0C to 91\u00b0C, \u0394complementG from \u221230 to \u221292 kcal/mol, \u0394homodimerG from \u22121.5 to \u221212 kcal/mol, and \u0394hairpinG from +1.8 to \u22126 kcal/mol. Prototype arrays were synthesized and hybridized to 4 \u03bcg of pure genomic DNA from one of the four species, as described below.In order to determine the effect of design parameters on probe sensitivity and specificity, we constructed a prototype array containing several hundred probes for each member of ten gene families having representatives in all four target species. For each target gene, probes were selected spanning a wide range of design parameters. Probe lengths ranged from 30 to 66 nt, GC content from 40% to 60%, predicted E. coli CFT073) was present in the hybridization mix, while DNA for another set of probes  was absent; thus the signal seen for E. faecalis probes is entirely due to non-specific hybridization and other sources of background noise. We found that probes with lengths above 50 nt gave significantly stronger signals, with better differentiation from background, than lengths in the 30 to 45 nt range. The predicted melting temperature and \u0394complementG are strongly correlated with probe length, but not entirely determined by it. We performed linear regression fits to the log intensity against each of the probe design parameters, and multiple regressions against several combinations of parameters. Of the individual probe parameters we examined, the best predictor of intensity  was \u0394complementG; the best multivariate predictor was a combination of \u0394complementG, \u0394homodimerG, and \u0394hairpinG.complementG is nonlinear, and shows evidence of chemical saturation for the most sensitive probes. In order to incorporate saturation into our probe response model and find the combination of thermodynamic parameters that was the best predictor of probe sensitivity, we fit our data to a Langmuir isotherm curve complementG, \u0394homodimerG, and \u0394hairpinG as follows:0a through 5.a We determined that a linear combination of the three free energies which we term \u201c\u0394adjustedG\u201d was the best predictor of hybridization intensity for probes complementary to the target DNA. The \u0394adjustedG is defined as:We observed that the relationship of log intensity to thermodynamic parameters such as \u0394mT\u226580\u00b0C, \u0394homodimerG>\u221212 kcal/mol, \u0394hairpinG>\u22126 kcal/mol, and \u0394adjustedG\u2264\u221255 kcal/mol.In subsequent array designs, we screened candidate probes to include only those with predicted k  across the PM sequence, and creating k mismatches at the location of the window. We generated random MM probes by selecting k random positions in the PM probe, with k\u200a=\u200a1, 2, 3, 6, 10, 15 or 20, and creating single MM at each position. In interval MM probes, mismatches were placed at regular intervals of size k, starting with a MM at the first base at the 5\u2032 end of the probe. MM probes with shifting PM regions were created for region lengths n between 15 and 30, and offset values s ranging from 2 to 29. For each combination of length and offset, a probe was generated by preserving a PM region of size n, starting at base position s, and creating a MM at every third base on either side of the PM region.We generated mismatch (MM) probes, derived from a selection of perfect match (PM) target probe sequences, in order to test the ability of probes designed against gene family members from one organism to detect orthologs with non-identical sequences from other organisms. Previous experiments suggested that hybridization to MM probes depends strongly not only on the number of mismatched bases, but also on their location and distribution across the length of the probe (data not shown). MM probes were generated using five different strategies, incorporating single, adjacent, random, interval, and shifting PM region mismatches. Single and adjacent MM probes were generated by sliding a window of size We generated 3,000 negative control probes, consisting of random sequences designed to have the same distribution of length and GC content as the target probes. BLAST searches of the random probes against the GenBank nt database showed that none had any perfect match alignments of length greater than 21 nt to any known sequence, so that none would be expected to hybridize to the organisms we tested on the arrays. These were used to determine background noise levels due to non-specific hybridization of target DNA and to fluorescence of the chip substrate.Arabidopsis calmodulin protein kinase 6 (CPK6) fiducial spots. The slides were hybridized with complementary CPK6-Cy3  and scanned to assure the quality of each array before hybridizing to DNA targets.DNA microarrays were prepared on glass microscope slides according to a photolabile deprotection strategy that has been previously described E. coli K12 MG1655, E. coli CFT073, E. faecalis V583 and S. aureus Mu50 were purchased from ATCC. The bacterial culture pellets were grown according to the instructions from ATCC and genomic DNA was extracted using the Epicentre DNA extraction kit according to the manufacturer's protocols. The DNA was quantified using a NanoDrop spectrophotometer . DNA samples were sonicated to fragment the DNA to a size range of 500\u20132000 bp, and then labeled using nick translation with Cy3-labeled random nonamer primers  and Klenow DNA polymerase at 37\u00b0C for 3 hr. The labeled DNA was precipitated in isopropanol, and the pellet was washed, dried, reconstituted and quantified. For each hybridization, 4 \u03bcg of labeled DNA was mixed with Cy3-labeled CPK6 oligomers, NimbleGen hybridization components and hybridization buffer according to the manufacturer's protocols. The arrays were hybridized with labeled DNA on a MAUI hybridization station  at 42\u00b0C for 16 hr. Arrays were washed with NimbleGen wash buffers I, II and III according to vendor protocols and scanned using an Axon GenePix 4000B scanner at 5 \u03bcm resolution.S. aureus DNA were added. S. aureus DNA was quantified using a Quant-iT PicoGreen ds DNA kit , serially diluted, and then spiked into 10 ng of aerosol sample DNA in quantities of 0.31 fg, 3.1 fg, 31 fg, 310 fg or 3.1 pg. We performed whole genome amplification of the combined samples  at 30\u00b0C for 16 hr using the REPLI-g whole genome amplification kit . The amplified material was inactivated at 65\u00b0C for 3 min and then purified using the QiaQuick PCR purification kit (Qiagen) to remove the primers and dNTPs. The entire amplified product was labeled with Cy3-random primer using the Klenow fragment and then hybridized to the array as described above.For limit of detection experiments, aerosol filters were kindly supplied by the BioWatch program and DNA was extracted using the MoBio UltraClean Soil DNA kit , as described in E.coli O157:H7 strain EDL933, Staphylococcus saprophyticus subsp. saprophyticus strain ATCC 15305, Salmonella enterica subspecies enterica serovar Paratyphi A strain ATCC 9150, and Streptococcus pyogenes strain MGAS5005 were purchased from ATCC. Samples were prepared from these organisms as described above for the other pure bacterial cultures.For experiments on detection of virulence gene orthologs in related organisms, 2-transformed intensities. Each probe on an array was considered to have a positive signal if the median log2 intensity of its technical replicates was above a detection threshold calculated for that array. The detection threshold was determined by using random control probes to model background noise. For each array in the target probe specificity and limit of detection experiments, the detection threshold was set to the median log2 intensity of the random control probes, plus 4 times the standard deviation of the log2 intensities. For detection of virulence gene orthologs in organisms other than the sources of probe target sequences, a more stringent threshold defined as the 99th percentile of the random control intensities was used.Data were analyzed using custom software based on the R programming environment and BioConductor packages. Each probe was randomly spotted in three to five replicates to control for positional effects on the array. Data from replicate probes were summarized by the median of the loghttp://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO series accession number GSE11010.The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus .To assess the ability of the NimbleGen array to reliably identify a target organism of known genome sequence, we performed BLAST searches for all target probe sequences against the four genomes (sequences derived from GenBank), and selected subsets of probes that had a full length perfect match to one genome, and no perfect match longer than 16 nt to any of the other 3 genomes. We refer to these probes as strain-specific probes. We performed hybridizations with purified genomic DNA from either E. coli K12 (data not shown). The true positive rate of detection, measured by the fraction of probes specific to the hybridized strain with intensity over the threshold (median+4 SD) is 100%. The false positive rate, the fraction of intensities over the threshold for probes specific to a different strain, was only 0.29%.In each case, the probes that were specific to virulence genes present in the target strain had much higher signal intensities than the random control probes and probes specific to the other three organisms. The same pattern was observed in the hybridization with E. coli) as two of the target strains; one is a different species of the same genus (Staphylococcus) as a target strain; one belongs to a different genus  of the same family as E.coli; and one (S. pyogenes) belongs to a different family of the same order as E.faecalis. All four strains were found by our HMM analysis to possess orthologs for a variety of virulence gene families; through the results of this analysis, we were able to divide the 160 gene families with probes on the array into \u201cpresent\u201d and \u201cabsent\u201d groups  in a given strain.In order to assess the ability of probes designed against gene family members from one organism to detect orthologs with non-identical sequences from other organisms, we performed two sets of experiments, one using the perfect match target probes, the other (to be discussed below) using the mismatch probes. In the first set of experiments, arrays were hybridized to DNA from four bacterial strains that do not have organism-specific probes on the array, as described in In the analysis of arrays from this experiment, a gene family was considered \u201cdetected\u201d if at least one probe specific for that family had median intensity above the detection threshold. A detailed listing of gene families, indicating the strains in which they are present and/or detected, is given in supporting information file E. coli O157:H7 strain EDL933, 159 of the 160 gene families were represented by an ortholog. All of them were detected; in addition, an efflux pump protein family not present in this strain was also detected, yielding 100% sensitivity and 0% specificity rates.In In the other bacterial strains tested, the specificity averaged 93.5%, and sensitivity ranged from 69% to 28%, decreasing with the taxonomic distance of the test strain from the closest strain with perfect match probes on the array.E. coli O157:H7, the false positive rate was 7.3% or less in all hybridizations performed.Our results from this small set of organisms suggest that probes designed according to our strategy against gene family members from one species can reliably detect orthologs in different species of the same genus, and even different genera of the same taxonomic family. Excepting the unusual case of To more comprehensively assess the factors influencing the balance between probe sensitivity and specificity, we analyzed data from two series of probes, containing single and multiple mismatches respectively. We first examined data from probes that perfectly matched the hybridized strain, except for a single mismatch (MM) base placed at a known position. We investigated the effect of the MM position on the probe intensity, relative to the intensity of the corresponding perfect match (PM) probe for the same hybridization. As shown in this figure, single MM probe intensities varied with the position of the MM. On NimbleGen arrays, the 3\u2032 end of the probe is attached by a 5-T linker to the glass surface of the array. We observed that mismatches located 7 to 20 nt from the 5\u2032 end of the probe had the strongest negative impact on hybridization, while mismatches located on one of the 12 nucleotides closest to the linker had virtually no discernable effect. We also note that, even at the position of maximum effect, 15 nt from the 5\u2032 end, single mismatches have relatively small impact, with only a 35% reduction of intensity relative to the corresponding PM probe. The reduction in intensity appears to be greater for shorter than for longer probes (data not shown).In the single-MM experiments, MM probes were generated containing all three possible choices of MM base at each position. We found no consistent difference in intensity between probes using the complement of the PM base and probes generated by transition or non-complementary transversion of the PM base.When probes contained multiple mismatches to the genome of the hybridized strain, we found that the reduction in intensity depended not only on the number of mismatched bases, but also on the length of the longest PM sequence between mismatches. Consequently, longer probes tend to be more tolerant of mismatches. The relationship between the number of MM bases, the longest PM region length, and the reduction in intensity relative to the cognate PM probe is shown in We observed that probes with two or three mismatches to the hybridized strain had at least half the intensity of the related PM probes, provided there was at least one PM region with length\u226529 nt. This was nearly always the case for 60-mer probes. Probes with six mismatches had greater signal reduction, but still had 30% or more of the PM probe intensity when the mismatches were clustered toward one end of the probe, leaving a 29 nt or longer PM region.Probes with 10 or more MM bases showed even greater signal reduction, and also more variability in reduction between probes. We conjecture that this variability is related to the position of the PM regions within the probe, with regions overlapping the 5\u2032 half of the probe having a stronger positive effect on signal intensity. Additional experiments using a wider variety of MM configurations would be required to test this hypothesis adequately.In general, all probes with PM regions\u226529 nt had intensities above the detection threshold. Probes with shorter maximal PM regions were detected some of the time, but not consistently.S. aureus DNA in amounts ranging from 0.31 fg to 3.1 pg, amplified, labeled and hybridized to arrays, as described in S. aureus chromosome has a mass of about 2.95 fg.Several experiments were performed to show the dynamic range and limit of detection of our array, along with its ability to identify specific organisms within a complex background, when combined with our protocol for sample preparation. We created six target microbial DNA samples using DNA isolated from an aerosol sample (24 hour filter collection from an urban environment) as complex background material. One target contained background DNA only; the others were spiked with fragmented complementG for arrays hybridized to each of the six samples. In the unspiked aerosol background DNA, we found only a few probes with signals barely above the detection threshold; therefore we expect that the signal seen in the spiked samples is mostly or entirely due to the added S. aureus DNA. With 0.31 fg of S. aureus DNA, we observe about 36% of S. aureus\u2013specific probes with signals above the threshold. The detectable probes cover about 37% of the targeted virulence gene orthologs. This level of detection was reproducibly observed in multiple experiments. With 3.1 fg, we see that 100% of the S. aureus specific probes were above the detection threshold. With 31, 310 or 3100 fg, virtually all of the S. aureus specific probes were saturated, with intensities within a factor of two below the maximum possible intensity.The emerging threat presented by novel pathogens, whether they arise naturally or are deliberately engineered, creates a need for detection systems that can warn public health authorities about a potential outbreak and help them select appropriate countermeasures. Ideally, such a system will be able to determine the virulence and antibiotic resistance mechanisms present in a sample of unidentified microorganisms, even when the sample includes organisms never previously encountered. As a step toward developing such a system, we have produced and tested a series of highly sensitive and specific functional gene arrays using the NimbleGen platform. These are the first functional gene arrays created that can quantify the presence or absence of hundreds of virulence gene families with a single assay. Our goals for this study were to develop methods for design of gene family-specific probes, to measure the sensitivity and specificity of these arrays, and to assess the validity of the FGA approach for detecting a broad spectrum of virulence and antibiotic resistance mechanisms in unknown as well as known microorganisms without culturing them.E. coli K12, E. coli CFT073, S. aureus and/or E. faecalis. These sequences were selected using a collection of over 700 hidden Markov models, each of which was trained against sequences of a single virulence or antibiotic resistance gene family, identified by an extensive literature search. Using these models, more than 200,000 targets were identified in a database of bacterial, viral and other genome sequences; the sequences with probes on the current array are those identified in one of the four target strains. While this set of models targets a substantial fraction of the virulence and antibiotic resistance gene families known at present, future arrays in this series will be based on a comprehensive set of over 1,500 HMMs covering the majority of known virulence and A/R related genes. .The array described in this report includes probes for 1,245 target sequences, within genes belonging to 160 virulence and antibiotic resistance gene families, identified in We used a novel approach to design groups of probes specific for virulence gene families. Prior to our study, there was no software available that could design minimal sets of family-specific probes for such a wide variety of sequences from unrelated organisms. Sequences within a gene family frequently are highly polymorphic at the nucleotide level, despite the functional conservation within the family. In order to minimize the total number of probes while covering as many families as possible across a phylogenetically diverse set of organisms, we developed rigorous algorithms to choose conserved probes that ensured detection of divergent sequences within a family.We note that the optimal characteristics of probes for gene family detection differ greatly from those for applications such as gene expression, in which mismatch bases are not tolerated and ideal probes produce linear signals in response to target concentration. For gene family detection, probes are required to tolerate a certain number of mismatches, commensurate with the degree of polymorphism within a gene family, without cross-hybridizing to members of other families. Linearity of response is not a concern, since our goal is to measure presence rather than abundance; in fact, we prefer to have probes that saturate in response to small quantities of complementary DNA. For this purpose, it worked well to calculate predicted free energies of hybridization for candidate probes against their complements, along with free energies for homodimer formation and self-hybridization. By setting a minimum threshold for an empirically derived linear combination of these free energies, we were able to select probes that had the necessary degree of sensitivity and mismatch tolerance.et al for comparative genome hybridization microarrays of cancer samples, they have shown that the amplification bias using bacteriophage Phi29 polymerase is less than 0.5% when sufficient material is used et al have reported that they were able to detect as little as 10 fg of microbial community DNA on their 50-mer functional gene array when combined with whole community genome amplification Because environmental samples may contain limited quantities of intact pathogen DNA, we expect that sample material will need to be amplified to generate the amount of target DNA required to produce a detectable signal on the array. Whole genome amplification has been used widely for bacterial genomes, producing high yields with low bias. In a study by Arriola S. aureus DNA as low as 0.31 fg and hybridized the amplified DNA to our virulence mechanism array. We found we were able to detect 100% of the virulence and antibiotic resistance probes expected to be present in S. aureus in a sample amplified from 3.1 fg of starting DNA. Since the S. aureus strain used has a genome mass of 2.95 fg, this starting amount is equivalent to slightly more than one genome copy.In our own limit of detection study, we applied whole genome amplification to initial quantities of fragmented et al showed that 50-mer probes, with 15-, 20- or 35-nt regions of PM to the hybridized genome sequence, had respective signal intensities approximately 1%, 4% or 50% of those obtained for perfect matches over the entire probe length et al reported that 70-mer probes, when hybridized to DNA with PM sequence lengths of 20, 35 or 50 nt, yielded signal intensities 10%, 32% or 55% respectively of those obtained for full length perfect matches We used two approaches to assess the array's tolerance for mismatches between probe and target sequence. The first was to design more than 24,000 probes with mismatch (MM) nucleotides at known positions, and compare their performance to perfect match (PM) probes targeting the same bacterial sequences. We found that long oligomer probes with 50 to 66 nucleotides yielded greater than 90% detection rates, even with up to three mismatches from the target sequence. Probes with larger numbers of mismatches still gave high detection rates, provided there was a region of PM sequence with length at least 29 nt. A previous study by Kane Higher MM tolerance was seen when the mismatches were placed nearer the 3\u2032 end of the probe, which is anchored to the array surface. Conversely, the region of maximum sensitivity to mismatches is about 1/3 of the distance from the 5\u2032 end to the 3\u2032 end. This position dependence is not accounted for in current algorithms for prediction of hybridization free energies. Our probe design software for future generations of virulence gene detection arrays will factor in this dependence, placing more highly conserved regions of gene families in the areas of maximum impact along the length of the probe.E. coli strain from the two used for probe design still gave good results, with 100% of gene families detected by one or more probes. A Salmonella strain, which belongs to the same taxonomic family (Enterobacteriaceae) as E. coli, also had a large fraction of expected gene families (69%) with detection signals. Interestingly, the array performed almost equally well with a member of the Staphylococcus genus, with 68% of expected gene families being detected. These results may simply indicate that taxonomic categories are only a rough indication of phylogenetic relatedness. These preliminary results are encouraging, at any rate; they suggest that a future array with probes for each gene family sampled intelligently from the whole range of bacterial taxonomic families stands a reasonable chance of being able to detect orthologs from species that are not currently sequenced.Our other approach to assess MM tolerance was to hybridize the array to genomic DNA from organisms with varying degrees of relatedness to the four target strains used for probe design. We found that a different One of the limitations of functional gene arrays is that they cannot detect SNP-based or small indel-based mutations that affect virulence or resistance, because probes are selected to not be sensitive to small mutations. This is an unavoidable cost of designing an array to detect broad patterns of gene presence. Thus, an ideal platform for pathogen detection would pair the broad virulence mechanism array with a resequencing array for specific virulence genes, whose polymorphisms have well understood effects on virulence or drug resistance. In this scenario, the resequencing array would be used as a secondary analysis if the broad mechanism array indicated the presence of virulence genes known to have important sequence variations.Finally, we emphasize the value of profiling multiple virulence related gene families in parallel, a unique advantage of microarray-based detection systems. Many virulence mechanisms require the coordinated actions of gene products from multiple gene families; therefore the presence in an organism of orthologs from most of the relevant families constitutes much stronger evidence for possession of a mechanism than the presence of one or two orthologs. We are developing analysis algorithms that will enable us to assign probabilities for the existence of particular virulence mechanisms in an unknown organism, using the unique discriminative power afforded by a multiple-family functional gene array.http://www.fas.org/sgp/crs/terror/RL32152.html), and to assess the pathogenic capabilities of unidentified organisms. Thus, our array can provide orthogonal confirmation for signature-based detection methods such as PCR. This array can also be used to differentiate virulent and avirulent strains by including antivirulence genes on the array The NimbleGen virulence gene array we developed shows great promise for detection of a broad range of virulence and antibiotic resistance genes. In addition to providing strain-level identification of known organisms, this technology will be valuable for functional characterization of unknown biothreat organisms. As a concrete example, we will use future versions of the array to identify or provide nearest-neighbor matches to organisms present in environmental samples collected by the BioWatch program .(0.09 MB XLS)Click here for additional data file."}
+{"text": "High-density oligonucleotide arrays are effective tools for genotyping numerous loci simultaneously. In small genome species (genome size: < ~300 Mb), whole-genome DNA hybridization to expression arrays has been used for various applications. In large genome species, transcript hybridization to expression arrays has been used for genotyping. Although rice is a fully sequenced model plant of medium genome size (~400 Mb), there are a few examples of the use of rice oligonucleotide array as a genotyping tool.\u00ae Rice Genome Array, using the rice cultivars with full genome sequence, japonica cultivar Nipponbare and indica cultivar 93-11. Both genomes were surveyed for all probe target sequences. Only completely matched 25-mer single copy probes of the Nipponbare genome were extracted, and SFPs between them and 93-11 sequences were predicted. We investigated optimum conditions for SFP detection in both whole genome and transcript hybridization using differences between perfect match and mismatch probe intensities of non-polymorphic targets, assuming that these differences are representative of those between mismatch and perfect targets. Several statistical methods of SFP detection by whole-genome hybridization were compared under the optimized conditions. Causes of false positives and negatives in SFP detection in both types of hybridization were investigated.We compared the single feature polymorphism (SFP) detection performance of whole-genome and transcript hybridizations using the Affymetrix GeneChipOryza sativa) can be effectively utilized for whole genome genotyping to conduct mutant mapping and analysis of quantitative traits such as gene expression levels.The optimizations allowed a more than 20% increase in true SFP detection in whole-genome hybridization and a large improvement of SFP detection performance in transcript hybridization. Significance analysis of the microarray for log-transformed raw intensities of PM probes gave the best performance in whole genome hybridization, and 22,936 true SFPs were detected with 23.58% false positives by whole genome hybridization. For transcript hybridization, stable SFP detection was achieved for highly expressed genes, and about 3,500 SFPs were detected at a high sensitivity (> 50%) in both shoot and young panicle transcripts. High SFP detection performances of both genome and transcript hybridizations indicated that microarrays of a complex genome (e.g., of  Arabidopsis (genome size: ~125 Mb) by bulk segregant analysis [Arabidopsis [Arabidopsis [Mycobacterium tuberculosis (genome size: ~4.4 Mb) [Xenopus (genome size: ~3.1 Gb) [High-density oligonucleotide arrays are currently the most widely used high-throughput technology for whole-genome gene expression studies. Array technology also makes it possible to genotype thousands of nucleotide polymorphisms (NPs) efficiently , and detanalysis ; quantitbidopsis ,8,9; andbidopsis , Arabidobidopsis , malariabidopsis , and in ~4.4 Mb) . However~4.4 Mb) , Xenopus~3.1 Gb) , and mai~3.1 Gb) , whole-g~3.1 Gb) ,18,19, m~3.1 Gb) , wheat (~3.1 Gb) , and cow~3.1 Gb) .\u00ae Rice Genome Array . A trial SFP detection using whole-genome hybridization by the rice array was reported by Kumar et al., and more than 70% SFP detection sensitivity at about 10% estimated FDR  was verified by sequencing probe targets [et al., andthey detected 1,208 SFPs, and 60 out of 62 predicted SFPs were verified by sequencing predicted SFP-containing amplicons [japonica cultivar Nipponbare, which has been sequenced by a BAC-by-BAC approach [indica cultivar 93-11, which has been sequenced by a shotgun approach [\u00ae probes were designed to target the coding and 3'-UT regions of japonica transcripts, 4 SNPs/kb were expected in the target region of the 93-11 genome on an average. The two fully sequenced rice strains provided us with an opportunity for detailed analyses of SFP detection efficiency.Rice is important as both a food and model plant for the grasses. The genome size of rice (389 Mb) is relatively small amongst crop species, but is larger than that of malaria mosquitoes, which have the largest genome used in successful studies of whole-genome hybridization. Affymetrix supplies a 3'-expression array for rice, the Affymetrix GeneChip targets . SFP detmplicons . Howeverapproach , and theapproach . Nucleotapproach . One of \u00ae array [First, we searched all probe target sequences in Nipponbare and 93-11 genomes and predicted SFPs between them. Second, we investigated optimized experimental conditions to detect these SFPs by whole-genome hybridization. Several statistical methods for SFP detection were compared using whole-genome hybridization data of Nipponbare and 93-11 cultivars. Effects of several background corrections were also examined for maximum SFP detection performance. Third, SFP detection efficiency by cRNA hybridization was also investigated by applying our recently proposed method for simultaneously detecting nucleotide and expression polymorphisms (SNEP) using the Affymetrix GeneChip\u00ae array . Finally\u00ae array was designed for 48,564 japonica transcripts and 1,260 indica transcripts. A transcript is represented as a probe set. A probe set is made up of several probe pairs  comprised of Perfect Match (PM) and Mismatch (MM) probes. MM probes were designed to represent the non-specific hybridization signal value. Several probes on this array had a possibility of cross-hybridization with other regions of the rice genome because the array probes were designed before the completion of rice genome sequencing efforts. Only 25-mer completely matched and single copy probes of the Nipponbare genome were extracted and used in this study. To select single copy completely matched probes, similarities of all 628,725 PM probe target sequences of the Affymetrix GeneChip\u00ae array were searched against the Nipponbare genome by BLASTN analysis  containing probes, SFP, for the 93-11 genome.The Affymetrix GeneChipArabidopsis. Thus, it is important to investigate conditions that maximize hybridization signal intensity differences between completely matched and NP containing targets of probes on the Affymetrix GeneChip\u00ae arrays. There are two types of probes on the Affymetrix GeneChip\u00ae arrays; PM and MM. Although the MM probes were designed with single complementary substitutions at the 13th base (midpoint) of each PM probe to represent non-specific hybridization signal values, many studies have indicated that some MM probe intensities are frequently higher than those of the corresponding PM probes (designated: MM > PM) [The rice genome, which is about 389 Mb in size , may genMM > PM) ,28. The 10 intensities of raw PM values were used to determine SFPs in each analysis. SFP detection performance was evaluated by sensitivity, ratio of the correctly called to the expected number of SFPs, the false-positive rate (FPR), and the ratio of the number of falsely called to total called SFPs. Receiver operating characteristic (ROC) curves with sensitivity plotted against FPR, are shown in Figure All Affymetrix protocols for hybridization, washing, and scanning have been standardized , and wer10 PM intensities within a probe set between two strains for cRNA hybridization, and it is also applicable for gDNA hybridization. The log10 intensities of raw PM values were used to determine SFPs in each analysis. SFP detection performance was evaluated by ROC curves , SAM , and SNEs Figure . Althougs Figure . To exams Figure . No methet al. [Considering the wide range of potential applications of detected SFPs, it is worthwhile to investigate the distribution of SFPs in the genome. The distribution of the number of unique probes, predicted SFPs, correctly and falsely called SFPs, and the sensitivity of SFP detection in a 1-Mb window are indicated in Figure et al. . Howeveret al. . Our cor10 intensities and subjected to SFP calls according to the SNEP procedure [et al., it is difficult to detect SFPs at low expression levels [10 intensities of PM probes were above 2.5 in both the shoot and young panicle. Subsequently, probe sets with a high expression level, where the median log10 intensity of a probe set was above 2.5 for both Nipponbare and 93-11 transcripts, were extracted from the unique probe sets. The performance of SNEP using PM intensities for calling SFPs was investigated by ROC curves of the shoot and young panicle  of Nipponbare and 93-11 cultivars were obtained according to Affymetrix standard protocols. Raw PM values of all probes were transformed to logrocedure . As repon levels . To invee Figure . Data of-6, were markedly decreased by addition of low expression genes from 51.0% to 6.9% and from 53.6% to 7.7%, respectively . In other words, intron-targeting probes were called as SFPs by SNEP when the expression level of a gene was different. Although 3,393 and 3,513 SFPs were correctly called from shoot and young panicle data, respectively, 2,812 SFPs were identical due to their similar expression profiles  was calculated by nearest-neighbor thermodynamic parameters [G > -26 kcal/mol, the SFP detection sensitivity was improved from 38.89% to 56.72% with a similar FPR .SFP detection performance of whole-genome hybridization was inferior to that of transcript hybridization Figure . Low genrameters . We founEven at a stringent threshold of SFP detection, significant false-positive calls existed and there was a minimum FRP limit on the ROC curve at around 0.1 Figure , 3. SourMany new applications of oligonucleotide arrays have been developed in recent years. In this study, we describe a method to seek optimal conditions for SFP detection by both genome and mRNA hybridizations using the differences between PM and MM probe signal intensities of completely matched targets. These optimizations greatly improve SFP detection performances in both whole-genome and transcript hybridizations. This simple method is applicable to any other Affymetrix arrays of any species. Especially, for large genome species, this method will be useful to evaluate the possibility of SFP detection.O. sativa Nipponbare and 93-11. Sensitivity (38.9%) and FPR (22.4%) of SFP detection by whole-genome hybridization was less than the reported sensitivity (57%) and FPR (13%) of SFP detection by genome hybridization of Arabidopsis [Arabidopsis protocols [Arabidopsis whole-genome hybridization experiments, and the rice optimal concentration was ~20 times lower than that of Arabidopsis, considering their genome sizes. Although a lower target concentration was used for rice whole-genome hybridization and single copy probes were selected for analysis, PM and MM probe intensities could not be sufficiently differentiated. These limitations affected SFP detection. The GC content of rice genes was found to be higher than that of Arabidopsis [\u00ae Arabidopsis ATH1 Genome Array for Arabidopsis whole-genome hybridization (data not shown).Under the optimized conditions, SFP detection performances in both genome and mRNA hybridizations were evaluated using the whole genome sequences of the two sequenced strains of  and FPR 2.4% of Srotocols , 40 \u03bcg ositivity .9% and Fbidopsis . Thus, bOur results from the whole-genome approach suggested that false-positive probes were clustered due to amplification polymorphism caused by nearby SNPs. Thus, these false positives can be used as genetic markers if there is a real NP within 200 bp around them.Using shoot and young panicle transcripts, higher SFP detection sensitivities (51% and 54%) were observed with a similar FPR of 21%. Comparing our previous results, a sensitivity of 65% and FPR of 10% for 1,901 probe sets from the canonical rice data of the young panicle , the SFP10-intensity differences in a set. On the other hand, several issues aside from NP, such as alternative splicing or gene duplication, lead to SFPs. Gene duplication can result from unequal crossing over in chromosomal duplication, the outcomes of which can be quite different. Difference in exon-intron structure between duplicated genes in the 93-11 genome could also lead to these misclassifications. Some false positive SFPs detected by transcript hybridizations were attributed to various forms of genetic diversity such as copy number of a gene and alternative splicing. In other words, SNEP detects differences not only in nucleotide sequences and expression levels of a gene, but also in the gross structure of a gene.Using sequence analysis, in the focused 41,525 probe sets, 17,912 probe sets were expected to have SFP probes in the 93-11 genome  is a promising application of SFPs. In eQTL studies of yeast, genotypes of segregants were determined by about 3,000 SFPs from gDNA hybridization, and gene expression levels were determined by another type of microarray with spotted PCR products of the genome -42. The \u00ae array analysis by bulk gDNA hybridization is a cost-effective option for mapping a gene by bulk segregant analysis or QTL extreme mapping, because the rice genome is more than 2.5 times larger than that of Arabidopsis and the required number of reads by a sequencer should be proportional to the genome size.Recent revolutionary developments in sequencing technologies have challenged microarray technologies ,46. HoweOryza sativa) can be effectively utilized for whole genome genotyping to conduct mutant mapping and analysis of quantitative traits such as gene expression levels.In this study, we describe a method to seek optimal conditions for SFP detection by both genome and mRNA hybridizations using the differences between PM and MM probe signal intensities of completely matched targets. The optimizations allowed a more than 20% increase in true SFP detection in whole-genome hybridization and a large improvement of SFP detection performance in transcript hybridization. Significance analysis of the microarray for log-transformed raw intensities of PM probes gave the best performance in whole genome hybridization, and 22,936 true SFPs were detected with 23.58% false positives by whole genome hybridization. For transcript hybridization, stable SFP detection was achieved for highly expressed genes, and about 3,500 SFPs were detected at a high sensitivity (> 50%) in both shoot and young panicle transcripts. High SFP detection performances of both genome and transcript hybridizations indicated that microarrays of a complex genome . Purified DNA (300 ng) was labeled according to Arabidopsis Protocols [\u00ae arrays according to the Affymetrix standard protocol for RNA [\u00ae arrays according to the manufacturer's guidelines [\u00ae arrays were scanned with an Affymetrix GeneChip\u00ae Scanner 3000, and raw CEL files were generated by the Affymetrix GeneChip\u00ae Operating Software version 1.3. To investigate SFP detection performances of the two rice cultivars, four and five biological replicates of the hybridization as well as data read were carried out for independent gDNA and transcript samples, respectively. These array data were submitted to the Gene Expression Omnibus at http://www.ncbi.nlm.nih.gov/geo/, GSE16341. Gene expression data from 2-cm long young panicles of the two rice subspecies could be obtained as GSE16265.gDNA from leaves of two rice subspecies, rotocols , and rea for RNA . Total Ridelines . The Aff10-transformed intensity value of each feature was extracted and subjected to data analysis for SFP calls using ANOVA, SAM in package \"siggenes\" [http://www.ism.ac.jp/~fujisawa/SNEP/. SNEP was originally developed for transcript hybridization; however, it can also be used for SFP detection by whole-genome hybridization after modification of the SNEP script. PM probe intensities were transformed to log10 values; these are randomly grouped together in 500 PM probes, and SFPs were called in each group. This script is available at the SNEP site. SFP detection by transcript hybridization was performed using SNEP, as described previously [For SFP detection by whole-genome hybridization, all statistical analyses were performed with the freely available statistical package R. The raw CEL intensity files were analyzed using a series of methods implemented by the software Bioconductor . Backgroiggenes\" , and SNEeviously .G) of a PM probe to a complete match target at a wash temperature of 50\u00b0C was calculated according to the values of the nearest-neighbor thermodynamic parameters for DNA [The binding stability , ones with highly expression (middle), and the sensitivity of SFP detection at p < 10-6 by SNEP (bottom) across Nipponbare genome in each 3-Mb segment.Click here for file"}
+{"text": "Over 60% of protein-coding genes in vertebrates express mRNAs that undergo alternative splicing. The resulting collection of transcript isoforms poses significant challenges for contemporary biological assays. For example, RT-PCR validation of gene expression microarray results may be unsuccessful if the two technologies target different splice variants. Effective use of sequence-based technologies requires knowledge of the specific splice variant(s) that are targeted. In addition, the critical roles of alternative splice forms in biological function and in disease suggest that assay results may be more informative if analyzed in the context of the targeted splice variant.A number of contemporary technologies are used for analyzing transcripts or proteins. To enable investigation of the impact of splice variation on the interpretation of data derived from those technologies, we have developed SpliceCenter. SpliceCenter is a suite of user-friendly, web-based applications that includes programs for analysis of RT-PCR primer/probe sets, effectors of RNAi, microarrays, and protein-targeting technologies. Both interactive and high-throughput implementations of the tools are provided. The interactive versions of SpliceCenter tools provide visualizations of a gene's alternative transcripts and probe target positions, enabling the user to identify which splice variants are or are not targeted. The high-throughput batch versions accept user query files and provide results in tabular form. When, for example, we used SpliceCenter's batch siRNA-Check to process the Cancer Genome Anatomy Project's large-scale shRNA library, we found that only 59% of the 50,766 shRNAs in the library target all known splice variants of the target gene, 32% target some but not all, and 9% do not target any currently annotated transcript. provides unique, user-friendly applications for assessing the impact of transcript variation on the design and interpretation of RT-PCR, RNAi, gene expression microarrays, antibody-based detection, and mass spectrometry proteomics. The tools are intended for use by bench biologists as well as bioinformaticists.SpliceCenter  Technologies commonly used by biologists to investigate gene function include quantitative RT-PCR (qRT-PCR) assays, RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), gene expression microarrays, and antibody-based protein assays. Each of those technologies targets a small nucleic or amino acid sequence that, preferably, is unique to a specific gene.More than 60% of protein-coding genes in vertebrates exhibit splice variation ,2. Alter\u2022 Which specific splice variants are targeted by my assay? What other splice variants exist?\u2022 Do the RT-PCR primers/probes that I plan to use to validate microarray expression results target the same splice variants as were targeted by the microarray platform?\u2022 Did an siRNA fail to mediate RNAi silencing of a gene because it did not target the dominant splice isoform?\u2022 Is there known splice variation in my gene of interest that affects the protein coding portion of the transcript? Does the antibody that I plan to use target all potential protein products?\u2022 Where could I place RT PCR primers to target all splice variants? Where could I place RT PCR primers to amplify one specific splice variant to the exclusion of others?\u2022 Do expression values from one microarray fail to correlate with values from another microarray because the probesets target different splice variants?Questions such as those are not motivated by a particular research focus on alternative splicing, but rather by the need to account for the impact of splice variation in almost every high-throughput biological study. In our laboratories, for example, we have experienced several such issues, including an siRNA that failed to target the dominant transcript in a particular cell line and RT PCR results that failed to correlate with microarray expression data because different splice forms were targeted .EGFr), a major target for cancer therapy, can be expressed in the transmembrane receptor form or as a soluble isoform that competes with the receptor for binding of ligand. Transcripts with variation in the untranslated regions (UTRs) may be differentially regulated and therefore exhibit differences in spatial or temporal expression patterns.In addition to the pragmatic motivations for evaluating the splice variants targeted by a given assay, there may also be scientific benefit. Alternate splice forms have been associated with tissue-specific gene functions, developmental processes, and disease states (notably cancer) ,5. Genese.g. alternative, constitutive, retained, internal), and EST evidence/tissue of origin data. Although some of those programs are excellent resources for alternative splicing data, none of them provide query features and output focused on, and tuned to, the need of bench biologists to understand the impact of alternative splicing on their sequence-based or hybridization-based assays. A comparison of SpliceCenter with the other available resources is summarized in Table Several publicly-accessible websites provide data and utilities for investigating alternative splicing: AceView , Alternae.g., in antibody-based binding assays and mass spectrometry). SpliceCenter applications provide the novel ability to cross-compare the technologies .To meet the needs of biologists as well as bioinformaticists, we have developed SpliceCenter, a comprehensive, rapid, user-friendly suite of web-based tools for identifying the alternative transcripts targeted by contemporary technologies. The SpliceCenter tools include: \"Primer-Check\" for evaluation of qRT-PCR primers/probes, siRNA-Check\" for analysis of RNAi effectors, \"Array-Check\" for analysis of mRNA expression microarray probes, and \"Peptide-Check\" for analysis of peptides (e.g. siRNA and PCR primer sequences).SpliceCenter was developed using infrastructure from our previously described SpliceMiner application  includinSpliceCenter applications are web-based Java programs (JSPs and servlets) that query two mysql databases: Evidence Viewer Database (EVDB) and Microarray Database. EVDB contains the complete coding transcripts from RefSeq(release 29) and GenBankrelease 165) that represent distinct splice variants.  were problematic for our previously developed sequence alignment functions. The new sequence search component makes use of the open source PrimerMatch [We needed to develop new sequence-alignment components for PCR-Check and siRNA-Check because the relatively short sequence lengths  is widely accepted as the gold standard for validation of microarray expression studies and is also widely used in its own right for accurate measurement of mRNA levels. However, the process of designing oligonucleotide primers for qRT-PCR requires that all existing transcript variants be considered. For example, if qRT-PCR primers and microarray probes target different splice variants, validation of expression results may be compromised. Primer-Check shows the splice variants of a gene and indicates the target locations of PCR primers specified by the user, thereby allowing rapid determination of which variants are and are not targeted. That information can help in the design or selection of primers or in diagnosing unexpected results. The Primer-Check application also includes the option to construct graphical results that indicate the positions of both PCR primers and microarray probes to evaluate whether the two target the same variants. The user enters the target gene and sequence of the oligonucleotides selected for use as primers and/or detection probes. Primer-Check returns a graphical display of the splice variants of the gene and indicates the target locations of the primers Figure . Common \u2022 Primer design and selection: Whether designing custom primers or selecting commercial ones, it is important to identify the splice variants that will be targeted. Primer-Check can be used to ensure that selected primer pairs hybridize to all variants  and to screen for possible cross-hybridizations.\u2022 Investigation of anomalous results: One potential reason for failure of RT-PCR primers is that they are not targeting the splice variant(s) present in the particular sample being analyzed. Primer-Check is useful for trouble-shooting RT-PCR primers that fail to provide the expected amplification product.\u2022 Validation of microarray data: As already noted, qRT-PCR is considered to be the gold standard for validation of microarray results. Primer-Check can display the target locations of PCR primers and probes and the target locations of microarray probes in a single graphical display that shows directly whether PCR primers and microarray probes do, in fact, target the same variants.SEC23IP (P125), a gene that showed discordance between microarray and qRT-PCR results in the Dallas study [216392_s_at, also targets two additional transcript variants (BC063800 and AB019435), missing the fourth. The second probe set, 209175_at, targets only one of those additional variants (BC063800), missing the other two. In contrast, the PCR primers and probes from Applied Biosystems target all four reported variants corresponding to SEC23IP, leading to discordance between the qRT-PCR and microarray results. Primer-Check can help diagnose or even avoid such problems.The effect of splice variation on validation of microarray expression data by qRT-PCR data is by no means hypothetical. A study by Dallas and colleagues  found thas study . Primer-via the RNA interference pathway. Selecting an siRNA sequence that effectively targets a gene is a complex task that requires in silico prediction of the ability of the siRNA to mediate cleavage of the targeted transcript(s) while avoiding partially homologous sequences of other genes. Databases of experimentally-validated siRNAs and several tools to aid in design are available [RNAi technologies based on exogenously administered siRNAs or shRNAs are used extensively to investigate gene function. For compactness in the following descriptions, we will often use the term \"siRNA\" to include all of the standard RNAi effector molecules. siRNAs mediate sequence-specific gene silencing through targeted cleavage of a transcript vailable ,19. To avia an intuitive graphical display. If an siRNA targets a sequence that occurs in more than one gene, multiple graphics panels, one for each gene, are displayed.\u2022 Selection or design of siRNA (or shRNA) sequences: Whether designing custom siRNAs or selecting commercial ones, it is important to understand which variants will be targeted. The siRNA-Check application can be used to confirm targeting of all variants or selective targeting of a particular variant . In interactive mode, the application identifies siRNA target sequences within a gene BAD and YWHAZ, we observed differential expression of some of the untargeted transcript variants [BAD (siBAD.1 and siBAD.3) mediated a significant decrease in mRNA levels when all variants of the gene were assayed . But siBAD.2 produced no knockdown. Transcript-specific qRT-PCR showed that NM_004322, the transcript variant targeted by siBAD.2, represents only 1% of BAD mRNA levels in the cell line studied. We saw analogous results for the gene YWHAZ  [see Additional file \u2022 Clarification of anomalous results: siRNA-Check provides a quick, easy way to investigate the possibility that failure to silence a gene is due to splice variation. To cite one example from our own work, when we were trying to knock down expression of two apoptosis-associated genes, variants . For exavariants , two siRTranscript expression microarrays are being used as tools throughout basic and clinical research. The results of microarray expression studies are usually reported as lists of over- or under-expressed genes. However, failure of the oligonucleotide probes to detect all splice variants of the target gene may confound interpretation of the results. For example, a recent analysis of gene expression data obtained for different microarray platforms showed that genes exhibiting transcript variation had a lower signal agreement between platforms than did genes with less alternative splicing . Array-CACP1 gene. Array-Check thus provides a quick means of performing a side-by-side comparison of the coverage of splice variants by microarray platforms. It can be used in the mining of historical microarray datasets to ensure that an older platform provided good coverage of all variants of the gene of interest. It may also be useful in selecting a platform for a new study. As shown in the figure, the U133A and U133 Plus 2 Affymetrix arrays provide better coverage of the ACP1 variants than does the U95A array.\u2022 Microarray platform evaluation: Figure ACP1.\u2022 Microarray platform correlation: Comparison of expression values from different microarray platforms is prone to misinterpretation if the platforms target different splice variants. Array-Check provides a mechanism for comparison of probe target locations to identify potential splicing-related differences. For example, Figure \u2022 Trouble-shooting anomalous results: Alternative splicing is a potential source of inconsistent expression measurements among the probes in a nominal probe set. Array-Check provides a rapid means for ascertaining the known variants that are targeted or missed by probes on a given microarray platform. Older microarrays were designed before the availability of detailed annotation of many of the recently-identified transcript variants. Array-Check indicates splice variant coverage in the context of up-to-date information on transcript variation.Alternative splicing plays a critical role in higher organisms by increasing the functional diversity of proteins. Isoforms that differ minimally in structure may perform very different functions or may perform the same function in different cell types or at different stages of development. Failure to take splice variation into account can lead to inaccurate or incorrect interpretation of experimental results. Mass spectrometry and antibody-binding assays, the most common technologies in proteomic research, are susceptible to such problems. For those technologies, Peptide-Check provides a simple interface that accepts one or more short peptide sequences, generates a visualization of the known splice variants of the source gene, and shows the location, within the mRNA transcript, of the nucleotide sequence that codes for the peptide. Common use-cases include the following:\u2022 Design and analysis of peptide immunogens and antigens: Peptide-Check has many applications in the context of technologies that use antibodies or other ligands that target peptide sequences. To cite one common example, animals are often immunized with a peptide to generate antibodies against a specific protein. Peptide-Check can assist in selecting an immunizing peptide that occurs in all splice forms of the protein or, conversely, in only one particular form. The latter type of specificity may be particularly useful for identification of the biological or pathological roles of individual protein isoforms. For example, antibodies raised against peptides that represent unique splice variants of p53 have helped to elucidate details of the molecule's tumor suppressor function . Peptide\u2022 Analysis of Mass spectrometry results: Mass spectrometry is increasingly being used to identify and/or quantify proteins in a biological sample after peptidolysis. The first step is to identify peptides on the basis of mass/charge ratios, partial sequences, and/or chromatographic elution times. The identity of the original protein is then inferred from the peptides by any of a number of available software packages (reviewed in ). Peptide.g. siRNA or shRNA). Two result files are produced: 1) the Hit/Miss Report and 2) the siRNA Position Report  or FireFox (version 2.0 or better) but are not aware of compatibility issues with other browsers.The SpliceCenter website is available for use by academic, government, or commercial users without restriction or charge. The address of the site is: MCR and JAC designed and implemented the SpliceCenter applications, with input by BRZ and JNW. ABK designed and implemented the EVDB database and EVDB build process. MCR drafted the manuscript, with extensive input by NJC, BRZ, and JNW. NJC contributed to the design of the siRNA-Check application. All authors gave approval of the final version to be published.This file contains the Microarray Database schema, and a use case comparison of a task performed either with SpliceMiner or with SpliceCenter.Click here for fileBAD. Note that siBAD.2 does not target NM_032989. (b) Graphical output from siRNA-Check for siRNAs corresponding to the YWHAZ gene. Note that siYHWAZ.1 targets NM_003406 but not NM_145690. See Martin, et al.[siRNA-Check. (a) Graphical output from siRNA-Check for siRNAs designed to target Click here for filePeptide-Check Graphical output from Peptide-Check for peptides designed to target isoforms of p53 Peptide 1: DQTSFQKENC \u2013 p53\u03b2, Peptide 2: MLLDLRWCYFLINSS \u2013 p53\u03b3 (See ,25 for fClick here for file"}
+{"text": "The use of microarray technology for describing changes in mRNA expression to address ecological and evolutionary questions is becoming increasingly popular. Since three-spine stickleback are an important ecological and evolutionary model-species as well as an emerging model for eco-toxicology, the ability to have a functional and flexible microarray platform for transcriptome studies will greatly enhance the research potential in these areas.We designed 43,392 unique oligonucleotide probes representing 19,274 genes , and tested the hybridization performance of both DNA and RNA from different populations to determine the efficacy of probe design for transcriptome analysis using the Agilent array platform. The majority of probes were functional as evidenced by the DNA hybridization success, and 30,946 probes  had a signal that was significantly above background for RNA isolated from liver tissue. Genes identified as being expressed in liver tissue were grouped into functional categories for each of the three Gene Ontology groups: biological process, molecular function, and cellular component. As expected, the highest proportions of functional categories belonged to those associated with metabolic functions: metabolic process, binding, catabolism, and organelles.The probe and microarray design presented here provides an important step facilitating transcriptomics research for this important research organism by providing a set of over 43,000 probes whose hybridization success and specificity to liver expression has been demonstrated. Probes can easily be added or removed from the current design to tailor the array to specific experiments and additional flexibility lies in the ability to perform either one-color or two-color hybridizations. Microarrays and other whole genome methods are increasingly being applied to examine transcription patterns relevant for ecology and evolution in wild populations e.g,2). One . One 2])Gasterosteus aculeatus) stems from its well-documented history of parallel episodes of colonization from marine habitats followed by population divergence [EDA and pixt1 [The evolutionary significance of three-spine stickleback . This array design accounts for differential splicing by creating transcript-specific probes whenever possible. Since these probes are created in situ probe creation process, probes are high quality and the background signal is quite low. However, due to unused space, there were 255 probes that were replicated on the array (7 of these are in triplicate). Of the replicated probes with a significant hybridization, there was almost perfect correlation among these probes  had an intensity value significantly above background in all individuals. Only two probes clearly did not hybridize as the signal from these probes was not significant in all but one individual. These two probes were designed from the same transcript, however two other transcripts from the same gene had significant signal intensities. Five additional probes had 1-3 individuals that were not significantly above background. The hybridization signal intensity was quite high for many probes for the DNA samples Figure , and aboFor liver tissue mRNA, 30,946 out of 43,654 probes (71%) had a signal intensity that was significantly above background for at least 15 out of 17 individuals. These probes represented 14,615 genes (75.8%). A more conservative threshold using probes flagged as \"WellAboveBG\" was also applied to these data. With this threshold, 25,213 probes  were considered significantly expressed in liver tissue. The proportion of significantly expressed genes is higher than observed in a study using a similar array system in mice, where 12, 845 (54.5%) of the genes surveyed were considered \"transcriptionally active\" [The distribution of signal intensity for RNA was much more varied than with DNA and many more features had low signal intensity Figure . This is. BLAST searches were also used to increase the annotation information. From Biomart, a human UniProt accession number was obtained if possible to be used for functional characterization since the stickleback genes are not sufficiently annotated with regard to function. Approximately 2,959 genes  which had a significant intensity were not assigned a human UniProt accession number.Three-spine stickleback genes that exhibited a signal well above background (15 of 17 arrays flagged IsWellAboveBG)  were examined for functional characterization. Genes were initially matched to their putative human orthologs using Biomart from Ensembl  and the human generic GO Slim categories. Genes were placed into the \"slimmed\" categories for biological process (BP), molecular function (MF), and cellular component (CC). There were 877 unannotated genes of the 8,912 that had UniProt accession numbers.Functional categories were assigned using Go Term Mapper The largest proportion of genes were involved in metabolism (BP: 54.3%), binding (MF: 73.5%), specifically protein binding (48.2%) and catabolism (MF: 36.7%). Other than cell (85.5%) and intracellular (70.5%), organelles were the most represented category of the cellular component (58.0%). These results are consistent with expectations as the liver plays an important role in metabolism, and these functions occur in organelles, mostly mitochondria.As further evidence that the mRNA expressed in this experiment are characteristic of liver tissue expression, we compared our results to the transcriptionally active genes from mouse liver tissue . Using Gin situ probe creation, there is low background which leads to a high degree of reproducibility between array batches. Approximately 43,000 probes, representing 19,274 genes, are now available for use for transcriptomics studies of three-spine stickleback. Probes can easily be added or removed from the current design to tailor the array to specific experiments as not all genes will be expressed in all tissues. Additional flexibility lies in the ability to perform either one-color or two-color hybridizations. Both types of hybridizations were used successfully in the testing of the arrays.The Agilent Gene Expression Array Platform provides an extremely flexible custom array format for creating arrays for expression analysis. Because of the RNA sampled from fish from three study populations were tested on the arrays: Helsinki , Lake Pulmanki  and Lake V\u00e4ttern . Full-sib F1 families were created by crossing parental fish at the sampling sites, and fertilized eggs were transported to the laboratory. Initially, the offspring were maintained with water at 17 \u00b1 1\u00b0C and a photoperiod of 18 h light 6 h dark, and six months after hatching the environmental conditions were gradually changed to complete darkness (24 h dark) and 9 \u00b1 1\u00b0C to simulate wintering conditions. After five months the environmental conditions were changed back to 18/6h L/D photoperiod, 17 \u00b1 1\u00b0C and new crosses were made to obtain F2 offspring from each population. At the time of the experiments the F2 offspring were approximately 20 months old. They were adult fish, although at the time of the experiment, reproductively inactive.Fish were sampled from the tanks and immediately euthanized with a lethal dose of MS-222 anesthetic. Livers were removed from fish immediately, frozen in liquid nitrogen, and stored at -80\u00b0C. Six fish per population were analyzed for RNA, but one array (from the Pulmanki population) was of poor quality and was removed from the analysis. Since fish were not reproductively active, it was impossible to determine sex before the treatment. Subsequent dissection and visualization of reproductive structures revealed that there were 4 females and 13 males in the experiment.In order to determine the functionality of the array, two representatives from each of the above populations and a fourth population, representing an evolutionarily divergent lineage, was used for testing DNA hybridization to the arrays. The two additional DNA samples are from two individuals from the Pacific Ocean lineage collected from Shiomi River, Japan . However, one array, containing individuals from Lake V\u00e4ttern and Lake Pulmanki, was removed from analysis due to poor quality.Microarrays were designed using the custom gene expression 4 \u00d7 44K platform from Agilent which consists of 4 arrays per slide with 45,220 features, 43,803 of which are user-defined 60-base pair (bp) oligonucleotide probes. It is a newer configuration of that which was previously described . There a. All known and novel transcripts (as defined by Ensembl)  were included initially for two separate probe design projects in eArray using the default options. Probes were designed using the Base Composition Methodology option in eArray which is an Agilent methodology which chooses probes based on empirically determined base-composition profiles. The option to choose probes biased towards the 3' end was selected as recommended by Agilent. With this option, the software attempts to design the probe within the first 1000 bases of the transcript. In the first project, two probes for each transcript were designed using the input transcripts as the 'genome' to prevent cross-hybridization. For the second project, all parameters were the same except stickleback EST sequences from NCBI were used as the 'genome'. The two different input 'genomes' were used in the attempt to detect other splicing variants that may not have been recognized in the genome database. The probe sets from the two projects were combined and lower quality probes (as assessed according to parameters in the methodology-rating 3 BC and 4 BC) were removed. Additionally, the majority of duplicate probes resulting from the two different design jobs were removed.Probes were designed in eArray 4.5 (Agilent) using known and novel transcripts from Ensembl Stickleback Assembly Broad S1, database version 42.1b There are 43,654 stickleback probes on the microarray, and of these, 43,392 are unique as some duplicated probes were retained. The majority of probes were within 500 bases of the 3' end (83.5%), 14.7% were between 501 and 1000 bases from the 3' end, and only 1.8% were greater than 1000 bps from the end. Additionally, 149 randomly chosen probes from the Agilent catalogue probe sets were used to fill the array. The initial intention was that these may be used as negative controls; however due to conserved gene sequence, stickleback RNA and DNA hybridized to some of the probes, so they were all removed from the analysis.DNA was extracted from stickleback liver tissue using the Qiagen DNA Easy Kit using RNaseA treatment. DNA was eluted 2 times using 50 \u03bcL of water each time. 750 ng of DNA was digested with 5 units of AluI and 5 units of RsaI for 2 hours at 37\u00b0C and followed by 20 min at 65\u00b0C. The digested DNA was examined on an agarose gel to assess the effectiveness of the digestion.DNA labelling, hybridizations, and scanning were performed by the Finnish DNA Microarray Centre, which is an Agilent certified service provider. The Agilent protocol for Oligonucleotide Array-Based CGH for Genomic DNA Analysis was performed. Briefly, after digestion, DNA samples were either Cy3 or Cy5 labeled with Agilent's Genomic DNA labelling kit following manufacturer's protocols. After labelling, the DNA concentrations and specific activity was checked using the Nanodrop ND-1000 (NanoDrop Technologies).The Cy3 labelled sample and the Cy5 labelled sample were hybridized together onto Agilent's Gene Expression 4 \u00d7 44K custom array designed for three-spine stickleback at 65\u00b0C for 24 hours using Agilent's Oligo aCGH/ChIP-Chip Hybridization Kit. Washes were conducted with Agilent's Oligo aCGH/ChIP-on-Chip Wash Buffer set using Agilent's Stabilization and Drying solution according to the protocol. Arrays were scanned with Agilent Technologies Scanner, model G2505B. Spot intensities and other quality control features were extracted with Agilent's Feature Extraction Software version 9.5.3.1.Total RNA was isolated by means of Tri Reagent (Sigma) using the manufacturer's protocol. RNA was treated with DNase (Promega), 2.5 units for 1-2 \u03bcg RNA, and re-isolated using Tri Reagent. RNA concentration was quantified using a Nanodrop ND-1000, and RNA quality was assessed using an Experion Automated Electrophoresis System (Bio-Rad).RNA labelling, hybridizations, and scanning were performed by the Finnish DNA Microarray Centre. Briefly, total RNA (400 ng) was amplified and Cy3-labeled with Agilent's Low RNA Input Linear Amplification Kit PLUS, One Color (Agilent) along with Agilent's One-Color RNA Spike-in Kit following the manufacturer's protocols. After the labelling, the cRNA was examined with the Nanodrop ND-1000 and the Experion Automated Electrophoresis System cRNA to assess the concentration and quality of the labelling. Each sample (1.65 \u03bcg) was hybridized to the custom designed stickleback array at 65\u00b0C overnight (17 h) using Agilent's GE Hybridization Kit. Washes were conducted as recommended by the manufacturer using Agilent's Gene Expression Wash Pack without any stabilization or drying solution. Arrays were scanned with Agilent Technologies Scanner, model G2505B. Spot intensities and other quality control features were extracted with Agilent's Feature Extraction Software version 9.5.3.1. Array experiments are available at ArrayExpress with the accession numbers E-MEXP-2304 and E-MEXP-2309.. The software also determines the features which should be kept in the analysis by flagging features that are significantly above background as determined by a two-sided t-test. Features which were flagged as being positive and significant (IsPosAndSignif) were retained as long as that feature was positive and significant in at least 15 of the 17 arrays. Likewise, a more rigorous threshold was applied by using the flag IsWellAboveBG, which first determines if the feature is significant (IsPosAndSignif) and then determines if the background-subtracted signal is greater than 2.6 times the background standard deviation for that feature (approximates a 99% CI). Arrays were normalized using quantile normalization [Array quality was assessed through the use of Agilent control features as well as spike-in controls (Agilent 1-Color Spike-in Kit for RNA experiment). Due to poor hybridization, one array from the RNA experiment and one array from the DNA experiment were removed from further analysis. Processed signals from the Feature Extraction Software (v 9.5.3.1) were used for the analysis. Agilent's Feature Extraction software automatically normalizes within arrays, subtracts the background, and flags any outlier spots, either due to saturation or non-uniformity. Further details can be obtained from the Feature Extraction user guide lization  from theEL designed the probes and the microarray, conducted the laboratory work (with the exception of RNA labelling and array hybridizations), performed the statistical analysis, and drafted the manuscript. JM and CP supervised the research and assisted with the manuscript preparation. All authors read and approved the final manuscript.STable 1 - Probe information and gene annotation. Information about the probes including: probe name (Agilent), probe sequence, transcript and gene Ensembl identifiers, flags to determine feature significance, RNA median intensity and interquartile range and DNA median intensity . For the flag information for IsPosAndSignif and WellAboveBG, \"1\" means that the feature met the flag criteria in 15 out of 17 arrays.Click here for fileSTable 2. Comparison of the functional categories of liver tissue mRNA expression in mouse and stickleback.Click here for file"}
+{"text": "Following the domestication of maize over the past \u223c10,000 years, breeders have exploited the extensive genetic diversity of this species to mold its phenotype to meet human needs. The extent of structural variation, including copy number variation (CNV) and presence/absence variation (PAV), which are thought to contribute to the extraordinary phenotypic diversity and plasticity of this important crop, have not been elucidated. Whole-genome, array-based, comparative genomic hybridization (CGH) revealed a level of structural diversity between the inbred lines B73 and Mo17 that is unprecedented among higher eukaryotes. A detailed analysis of altered segments of DNA conservatively estimates that there are several hundred CNV sequences among the two genotypes, as well as several thousand PAV sequences that are present in B73 but not Mo17. Haplotype-specific PAVs contain hundreds of single-copy, expressed genes that may contribute to heterosis and to the extraordinary phenotypic diversity of this important crop. There is a growing appreciation for the role of genome structural variation in creating phenotypic variation within a species. Comparative genomic hybridization was used to compare the genome structures of two maize inbred lines, B73 and Mo17. The data reinforce the view that maize is a highly polymorphic species, but also show that there are often large genomic regions that have little or no variation. We identify several hundred sequences that, while present in both B73 and Mo17, have copy number differences in the two genomes. In addition, there are several thousand sequences, including at least 180 sequences annotated as single-copy genes, that are present in one genome but entirely missing in the other genome. This genome content variation leads to differences in transcript content between inbred lines and likely contributes to phenotypic diversity and heterosis in maize. Although many analyses of genetic variation have focused on single nucleotide polymorphisms (SNPs), there is a growing appreciation for the roles of structural variation as a cause for phenotypic variation Maize is phenotypically diverse There is also cytogenetic evidence for structural variation in the genomes of maize inbreds. Structural genomic variation involves alterations in DNA sequence beyond SNPs or small IDPs, and includes large-scale differences in chromosomal structure, altered locations of genes or repetitive elements, copy number variation (CNV) and presence/absence differences among haplotypes. Large-scale differences in chromosomal structure between maize inbred lines were first identified through cytogenetic studies. Barbara McClintock and others analyzed heterochromatic knob  content and size to characterize genome variation bz1 gene from eight different inbred lines revealed two significant findings Helitron transposons bz1 locus, such a genome typically contained one or more copies of these genes (or gene fragments) elsewhere. Second, comparison of multiple haplotypes revealed major differences in the amount and types of repetitive elements between genes. The same gene can be flanked by very different repetitive elements in different inbred lines a1-sh2 interval Sequence-based methodologies have documented structural diversity at a higher resolution . A high-density (2.1 million feature) oligonucleotide microarray was designed using the sequences of B73 BACs. Probes range in sizes from 45\u201385 bp were selected using slightly relaxed criteria (due to the overlap of adjacent BAC sequences and lack of assembly at the time of design) relative to those traditionally used for CGH probe design. The 2.12M probes were aligned to the B73 RefGen_v1 All of these perfectly matched probes were classified based on their repetitiveness and locations relative to predicted genes see ; Table 1Analyses of these regions were used to evaluate the quality of our genome-wide probe annotation. Several tracks in All probes were designed based on the B73 haplotype. To determine whether probe sequences were conserved in Mo17, probe sequences were aligned to a collection of 42,206,644 Mo17 whole-genome shotgun (WGS) reads generated by the DOE's Joint Genome Institute (JGI) and provided to us prior to publication by the Rohksar group. Based on these alignments each probe was classified as being a perfect match (100% identity and coverage), highly conserved (>97% identity and coverage), conserved (>90% identity and coverage), poorly conserved (>75% identity and >70% coverage) or as having no significant match in the JGI Mo17 data set. Over 80% of the probes were at least 90% identical to Mo17 sequences with over 90% of probe sequence coverage .The analysis of the four regions that have complete coverage of the Mo17 haplotype permitted us to compare the results of our genome-wide classifications with actual alignments of complete B73 and Mo17 sequences. Because the JGI collection of Mo17 WGS reads provides approximately 4\u00d7 coverage of the genome we expect some probes to be mis-classified as poorly conserved or as having no match in Mo17 simply due to incomplete sampling of the Mo17 genome. Overall, there was strong agreement between our classification of probes based on alignments to the Mo17 WGS reads and the genomic alignments shown in B73 and Mo17 genomic DNA samples were hybridized to the microarray using dye swaps as well as technical replication . AnalysiTo understand the biological causes of differences in hybridization signals between B73 and Mo17 we initially focused on the four regions shown in There are at least three biological reasons why a probe exhibits significant differences in signal after being hybridized to genomic DNA from two inbred lines. First, the probe sequence may have polymorphisms in the two genotypes (SNPs and IDPs). Second, the copy number of the probe in the genomic DNA might be different in the two genotypes being compared (CNV). Third, the probe sequence may be present in the genomic DNA of the reference genotype but not the other (PAV). It is important to remember that while all three reasons could explain why a probe would have a higher signal in B73 than in Mo17, only the second reason is likely to cause probes to have higher signals in Mo17 than in B73 because all probes were designed based on the B73 sequence.2(Mo17/B73) in probes with different levels of polymorphism between B73 and Mo17 (2(Mo17/B73) is zero. As the number of polymorphisms between B73 and Mo17 increases, the log2(Mo17/B73) value decreases and the percentage of probes that exhibit statistically significant differences in signal strength (q<0.0001) increases. Most of the probes with significant variation (68%) have 5 or more SNPs . Overall, this finding indicates that the majority of the significant differences in hybridization signals are due to the presence of multiple polymorphisms within the \u223c70 bp probe sequence or due to sequences that encompass or overlap the probe sequence that are present in B73 but absent from the Mo17 genome.The impact of sequence polymorphisms on hybridization can be observed by comparing the average logand Mo17 . For pro2(Mo17/B73) signals relative to the four B73/Mo17 haplotype alignments . This \u201cpermissive\u201d gene set  includes low-copy transposons as well as pseudogenes. The B73>Mo17 probes exhibit a distribution of genic and intergenic matches that is very similar to all probes. Interestingly, the Mo17>B73 probes are slightly depleted for intergenic probes and show an enrichment for probes near or within genes (Probes were compared to the full \u201cworking set\u201d of genes predicted by the MGSP (in genes . A very 2(Mo17/B73) signals for each probe were plotted relative to the genomic localization of the probes. As noted above, the majority of probes with significant B73>Mo17 hybridization detect structural variation. The genomic view provided in The probes were aligned to the B73 RefGen_v1 to visualize the patterns of structural variation along the B73 and Mo17 chromosomes . It shouThere are a number of highly conserved genomic regions that have very little or no structural variation between B73 and Mo17 . For exaWe performed further characterization of the large regions on chromosomes 1 and 8 with low rates of structural variation. The majority of probes within these regions (83%) are 100% conserved in B73 and Mo17 suggesting that these are low diversity regions. None of 388 primer pairs designed to amplify sequences within these regions revealed sequence variation between B73 and Mo17 that could be detected via agarose gel electrophoresis. In comparison, 13% of all primer sets designed for random genomics sites detect variation. We then used Temperature Gradient Capillary Electrophoresis (TGCE) to test whether B73 and Mo17 amplification products from 156 of the 388 primer pairs from the conserved regions contain SNPs or small IDPs. TGCE is sensitive enough to detect a single SNP in amplicons of over 800 bp and 1 bp IDPs in amplicons of \u223c500 bp There is a tendency for these large low diversity regions to be located near the central portions of the chromosomes and the centromere to be located near one side of a low diversity region for all chromosomes except 9. However, there are many low diversity regions that are not centromeric . These low diversity regions are likely to represent regions in which B73 and Mo17 are identical by descent or regions with no structural variation in the maize species. These low diversity regions also exhibit very low levels of differential gene expression. Only three of the 196 genes from the MGSP filtered gene set that are located in the conserved chromosome 1 or chromosome 8 regions and queried by the Affymetrix 17K microarray exhibit evidence for differential expression in seedling, embryo or endosperm tissue from B73 and Mo17 One visually striking feature in Based on the filtered gene set from the MGSP there are 31 genes within the \u223c2.6 Mb B73-specific interval. RNA-seq experiments provide evidence for expression of 14/31 genes located within this interval in B73 shoot apical meristem tissue  suggesting that many of the genes in this interval may be functional in B73. In addition, three of the genes within this interval are detected by the Affymetrix 17K microarray. The expression of all three of these genes are detected in B73 but not in Mo17 The sequence of the B73-specific region does not exhibit similarity to the chloroplast or mitochondrial genomes. The genes present within this region do not shown synteny to any specific region of the rice genome but are found scattered across different rice chromosomes. Maize chromosome 6 is syntenic to rice chromosomes 5 and 6 2(Mo17/B73) values for each segment was well approximated by a normal mixture model with four components, each corresponding to a different class of segments (2(Mo17/B73) value . As seen in the analysis of several well-annotated BACs, there are probes every \u223c400 bp in low-copy genomic DNA . CNVs casegments . Because3) value . Using s3) value . The DNA3) value .The B73>Mo17 and Mo17>B73 segments represent DNA sequences that are variable between B73 and Mo17. B73>Mo17 DNA segments could be the result of CNV or differences in genomic content (PAV) between the two lines. In an attempt to distinguish between these two possibilities we determined the proportion of each segment that was non-repetitive and that could be aligned to Mo17 WGS sequence reads. If a large proportion of the segment was found in Mo17 then it is likely that the segment is a CNV, while segments that are missing from the Mo17 WGS likely represent PAVs. The distribution of Mo17 coverage was very different for B73>Mo17 segments compared to the other categories of segments . Over 50The segments from the middle two distributions in The segment analysis identified a large number of DNA segments with variation in B73 and Mo17. There are 60 stringent Mo17>B73_CNV segments that are predicted to occur in more copies in Mo17 than in B73. There are 3,681 stringent B73>Mo17 segments including 356 segments that are CNVs and another 1,783 PAV segments that are putative examples of genome content variation.Several different approaches were used to validate the structural variants identified in this study. The 1,783 stringent B73>Mo17_PAV segments are predicted to be present in the B73 genome but absent from the Mo17 genome. Over 20,000 primer pairs were designed  and used to perform amplification from B73 and Mo17 genomic DNA. The numbers of primer pairs within each class of segment were determined . The proThe CGH analysis identified hundreds of candidate CNVs and thousands of PAVs. These sequences are spread throughout all ten of the maize chromosomes . The filThere is wide-spread appreciation for the high level of diversity within the maize species It is tempting to assume that all genomic regions are different in these two lines. However, by assessing the levels of variation along the B73 RefGen_v1 it quickly becomes obvious that this variation is not randomly distributed. We identified a number of large regions (>1 Mb) that have little or no variation. The fact that these regions co-localized with chromosomal regions that lack genetic markers that exhibit polymorphisms in the Intermated B73xMo17 (IBM) mapping population y1tb1Several groups have assessed molecular diversity in maize populations in studies designed to identify the targets of domestication and/or selection in maize We have identified thousands of examples of structural variation between the B73 and Mo17 genomes. The term structural variant is used to describe both sequences that are present in both individuals but have different copy numbers  and sequences that are present in one individual but absent in another . The unknown order and orientation of intra-BAC DNA sequence contigs will potentially lead to an over-estimation of the number of structural variation events by splitting some events into two different segments. However, it will also lead to an under-estimation of the number of events due to the fact that some smaller structural variant events which will not have enough sequence or probes on either side of a contig border to be called. We found that the 3,789 stringent B73>Mo17 or Mo17>B73 structural variants represent a minimum of 2,056 unique events .E. coli genome Differing probe densities, algorithms and statistical criteria complicate comparisons of rates of structural variation among organisms bz1 locus This study identified >400 putative CNVs between B73 and Mo17. A combination of genome homology searches and qPCR suggests that many of these sequences represent actual CNVs. There is evidence that these CNV can be the result of tandem duplications or duplications dispersed throughout the genome. There are a large number of tandem duplications in the maize genome Previous studies have suggested a high rate of near identical paralogs (NIPs) in the maize genome p1, c2, and r1 loci that exhibit epigenetic regulation b1 gene that controls expression and paramutation Many maize alleles that are known to be epigenetically regulated exhibit allelic variation for tandem repeats. There is allelic variation in the tandem duplication of coding regions at the In addition to the hundreds of CNV detected between B73 and Mo17 we also noted thousands of sequences that account for over 20 Mb of DNA that are present in the B73 genome and absent in the Mo17 genome. It is quite unexpected to find such a large number of sequences that are present within one haplotype of a species and missing from another. These include extreme examples such as the 2 Mb region on chromosome 6 as well as many smaller B73>Mo17_PAV sequences. Following the initial discovery of PAV sequences we sought to determine whether these PAVs included genes and to estimate the number of genes affected by PAV. Many PAV segments include genes contained within the MGSP filtered gene set. It is important to note that the MGSP filtered gene set was rigorously filtered to remove gene fragments and sequences with homology to transposable elements It is, however, difficult to determine the exact number of genes affected by PAV because this number is strongly influenced by the stringency used to identify PAV sequences and by the criteria used to identify genes within the PAV sequences. Using quite strict criteria for identifying segments and genes within the segments, the PAVs include 180 genes from the MGSP's filtered gene set  and the These present-absent sequences are spread throughout the B73 genome. These events differ in a significant way from those observed by Fu and Dooner The high level of PAV sequences between B73 and Mo17 may reflect ancient haplotype variation or more recent genomic rearrangements. We assessed the prevalence of 85 B73>Mo17_PAV segments in 22 other inbred lines  has pronounced and widespread effects on many traits. The high frequency of genome content differences suggests a large number of linked content differences. In this study we identify several thousand sequences that are present in B73 but missing in Mo17. If we assume that there are an equivalent number of sequences that are present in Mo17 but absent in B73 we would expect nearly 4,000 genome content differences distributed throughout the B73 and Mo17 genomes. The finding of single-copy, expressed PAVs among maize inbreds demonstrate that it will be important to obtain the genome sequences of a number of inbred lines to identify the full complement of genes present within the maize species. The large number of potential combinations of PAV sequences in hybrids also provides the opportunity for novel gene complements in hybrids relative to the parental lines. Previous analyses of gene expression in B73, Mo17 and the Fhttp://www.sanger.ac.uk/Software/analysis/SSAHA/) with a step-size of 1, nmer-size of 12 and a minimum match length of 33 bp. Up to five insertions/deletions were allowed in each match. Probes with <\u200a=\u200a15 close matches in the genome were included in the array design. Median final probe spacing was 450 bp. It should be noted that this set of probes was designed to facilitate sequence capture An oligonucleotide microarray was designed by Roche NimbleGen to perform comparative genomic hybridization (CGH) of maize inbreds (Copies of this design may be acquired by ordering: 080418_zea_mays_B73_CGH_HX1). A set of 14,423 maize BACs (downloaded March 2008) was used to design isothermal probes, varying in length from 45 bp to 85 bp and with a target Tm of 76C at a fixed interval of 50 bp. Probe sequences were repeat-masked by calculating the average 14-mer frequency for each probe, based on a frequency table generated from the complete set of BAC sequences available as of that date, and removing probes with an average 14-mer frequency higher than 400. Probe uniqueness was determined by comparing each probe to B73 RefGen_v1, using SSAHA (http://magi.plantgenomics.iastate.edu/) but did not meet the criteria for designation as multi-copy or icicle probes. Generally, the different classes of repetitive probes exhibit similar behavior and we will therefore refer to multi-copy, icicle and cereal repeat probes as \u201crepetitive probes\u201d. The remaining 1,461,771 probes were designated as non-repetitive. The location of each probe relative to genic sequences was determined through comparisons to gene models provided by the MGSP and were assigned to the following classes: exon, exon-intron (crosses exon/intron border), intron, 5\u2032 , 3\u2032 , intergenic (more than 2 kb from nearest gene). The conservation of sequences of individual probes to the Mo17 genome was classified via alignments to the 42,206,664 Mo17 WGS sequences provided by Daniel Rohskar from the DOE's Joint Genome Institute. Each probe was classified as perfect match (100% identity and coverage), highly conserved , conserved  or no match .The sequences of CGH probes were aligned to the B73 RefGen_v1 ; >90% of the probes  could be mapped with 100% identity and coverage . These iTotal genomic DNA isolated from two-week-old etiolated seedlings of maize inbreds B73 and Mo17 were labeled and hybridized following the methods described in Selzer et al. A segmentation analysis was performed using DNAcopy http://www.maizesequence.org) with the short read aligner NOVOALIGN (http://www.novocraft.com) using 32 bases. The low quality bases located at the end of reads were trimmed off by the program and only reads that mapped uniquely to the genome with a maximum of two mismatches including insertion/deletion (indel) across 32 bases were used for subsequent analyses. The reads uniquely mapped to genome were projected to gene models (release 4a.53).Gene expression information was obtained from several different sources. The Affymetrix data was obtained from 11-day old seedlings Click here for additional data file.Figure S2Significant hybridization differences are due to structural variation. The B73 and Mo17 sequences for two portions (A and B) of the bz1 locus  were aligned using Vista  which displays the percent identity as a sliding window of 100 bp (y-axis is 50% to 100% identity). The location of genes  and repeat elements  are shown above the VISTA alignment. The log2 is shown for each probe in this region. The red probes exhibit significantly different (q<0.0001) signal in B73 and Mo17. The repetitive annotation is shown as a track below the log signal (blue are repetitive probes and black are non-repetitive probes). The blue line indicates a segment with altered hybridization that was identified using DNAcopy. Note that these annotations are based on the genome-wide analysis, not detailed analyses of these regions. The last four probes in (A) and the fist four probes in (B) occur in regions where Mo17 does not have similar sequence at the allelic position but do not show significant differences in hybridization. This suggests that there are examples of sequences that are present in Mo17 but at a non-allelic position.(0.45 MB PPT)Click here for additional data file.Figure S3Density plots of sample chip signal intensity before and after global q-spline normalization. The distribution of B73 (red) and Mo17 (green) signals in raw data (A). Note that the distribution of signals is quite different for the two genotypes. In (B) the raw data were normalized using the global q-spline approach. This approach altered the distribution of signals such that the two genotypes exhibit similar distributions. In (C), the data were normalized using the B\u200a=\u200aM probes as a training set prior to global q-spline normalization. This approach preserves the original distributions of the signals for the two genotypes.(0.07 MB PPT)Click here for additional data file.Figure S4Alterations of the distribution of log2(M/B) values following different normalization approaches. In (A) a global q-spline normalization was applied to the data. The resulting log2(M/B) values exhibit a non-uniform distribution that is centered at 0.3 and a long tail towards negative log2(M/B) values. However, when the \u201cB\u200a=\u200aM\u201d probes are used as a training set prior to normalization, the distribution of values is centered near zero. This suggests the using the \u201cB\u200a=\u200aM\u2019 probes can provide a mechanism for appropriate normalization of this dataset.(0.06 MB PPT)Click here for additional data file.Figure S5Distribution of hybridization values in B73 and Mo17. (A) A volcano plot was used to show the distribution of q values (y-axis) relative to the log2(Mo17/B73) ratios (x-axis). Note that there are more significant probes with a negative log2 (M/B) value (upper left) than probes with a positive log2 (M>B) value. (B) The MA plot shows that there is a substantial bias towards low signal probes with a -M value.(0.07 MB PPT)Click here for additional data file.Figure S6Volcano and MA plots for classes of repetitive probes. (A) The multi-copy repeat probes (at least 5 copies of >97% identity and coverage) are shown in blue. Many of these probes have high hybridization signals. (B) The crosshyb repeat probes (at least five genomic loci with 90% identity and coverage) are shown in red. These probes rarely show significant differences and have a range of different hybridization values. (C) The cereal repeat probes  are shown in yellow.(0.38 MB PPT)Click here for additional data file.Figure S7Repetitive probes rarely report variation in B73 and Mo17. The chromosomal distribution (x-axis) is shown for each class of repetitive probe relative to the log2(Mo17/B73) (y-axis). (A) The multi-copy repeat probes (at least 5 copies of >97% identity and coverage) are shown in blue and all other probes are shown in gray. (B) The crosshyb repeat probes (at least copies that have 90% identity and coverage) are shown in red and all other probes are shown in gray. (C) The cereal repeat probes  are shown in yellow and all other probes are shown in gray.(0.54 MB PPT)Click here for additional data file.Figure S8Annotation of probes that exhibit significant (q<0.0001) variation in hybridization to B73 and Mo17 genomic DNA. (A) The percentage of all probes, B73>Mo17 probes and Mo17>B73 probes that are classified as non-repeat, multi-copy, icicle or cereal repeats. (B) For the same sets of probes, the conservation of probe sequence in Mo17 was assessed. (C) The location of significant probes relative to the MGSC working set of genes was also assessed. Each probe was classified as exon, other genic , or non-genic.(0.10 MB PPT)Click here for additional data file.Figure S9Volcano and MA plots for differing levels of conservation in Mo17 sequence. Each probe was compared to the Mo17 454 WGS sequence (provided by the Joint Genome Institute) and classified as perfect match (100% identity and coverage), highly conserved (>97% identity and coverage), conserved (>90% identity and coverage), poorly conserved (>75% identity and 70% coverage) or no match. The distribution of signals and variation for each type of probe are shown using volcano plots and MA plots. The pie chart shows the relative proportion of each type of probe.(0.50 MB PPT)Click here for additional data file.Figure S10Rates of variation and chromosomal distribution of probes with different levels of B73-Mo17 sequence conservation. The boxes indicate the positions of the centromeres Click here for additional data file.Figure S11Genomic regions of low (A) or high (B) levels of structural variation. The log2(Mo17/B73) hybridization intensities are plotted for a region on chromosome 8 (A) with low levels of probes that detect structural variation. In (B) the hybridization intensities are plotted for all of the probes within a region on chromosome 6 with high levels of probes that detect structural variation.(0.15 MB PPT)Click here for additional data file.Figure S12Annotation of probes that are within stringent segments that are present only in B73, or are higher in copy number in B73 or in Mo17. (A) The proportion of probes within stringent segments that are classified as non-repeat, multi-copy, icicle or cereal repeats. (B) For the same sets of probes, the conservation of probe sequence in Mo17 was assessed. (C) The location of probes relative to genes was also assessed. Each probe was classified as exon, exon-intron, intron, 5\u2032 2000bp or 3\u2032 2000bp.(0.19 MB PPT)Click here for additional data file.Figure S13Distribution of Mo17 coverage for DNA segments in each category. The proportion of stringent segments with the specified coverage by the Mo17 454 WGS reads are specified for each category. Note that the coverage statistics are the proportion of non-repetitive bases within the DNA sequence that are covered by Mo17 WGS sequence.(4.44 MB PPT)Click here for additional data file.Table S1IDPs within B73-specific chromosome 6 region.(0.04 MB XLS)Click here for additional data file.Table S2qPCR validation of Class 4 CNVs.(0.03 MB XLS)Click here for additional data file.Table S3Number of genes included within CNV and PAV segments.(0.03 MB XLS)Click here for additional data file.Table S4Annotation of 180 filtered genes located within PAV segments.(0.08 MB XLS)Click here for additional data file."}
+{"text": "A complete gene-expression microarray should preferably detect all genomic sequences that can be expressed as RNA in an organism, i.e. the transcriptome. However, our knowledge of a transcriptome of any organism still is incomplete and transcriptome information is continuously being updated. Here, we present a strategy to integrate heterogeneous sequence information that can be used as input for an up-to-date microarray design.Our algorithm consists of four steps. In the first step transcripts from different resources are grouped into Transcription Clusters (TCs) by looking at the similarity of all transcripts. TCs are groups of transcripts with a similar length. If a transcript is much smaller than a TC to which it is highly similar, it will be annotated as a subsequence of that TC and is used for probe design only if the probe designed for the TC does not query the subsequence. Secondly, all TCs are mapped to a genome assembly and gene information is added to the design. Thirdly TC members are ranked according to their trustworthiness and the most reliable sequence is used for the probe design. The last step is the actual array design. We have used this strategy to build an up-to-date zebrafish microarray.With our strategy and the software developed, it is possible to use a set of heterogeneous transcript resources for microarray design, reduce the number of candidate target sequences on which the design is based and reduce redundancy. By changing the parameters in the procedure it is possible to control the similarity within the TCs and thus the amount of candidate sequences for the design. The annotation of the microarray is carried out simultaneously with the design. The best scientific experiments are the ones based on the most recent scientific knowledge. Thus, in expression studies, our detector, i.e. the microarray, preferably would be based on the most recent and complete understanding of the genome and transcriptome. Although the annotation of commercially available microarrays is or can be ,2 updateThe concept of a gene has evolved from a stretch on the genome that encodes one protein to an entity that represents many and complex relations that exist between sequence and biological function. The definition of a gene by Gerstein et al. ,8 as 'a in silico predict genes and transcripts [On a technical level and fuelled by the information from next-generation sequencing experiments, we experience an unremitting flow of new transcription evidence and genome information. This data is used to improve the information in the transcriptome and genome repositories, such as Vega  and Ensenscripts -13. Morehttp://zfin.org[Orthogonal to the genome and transcriptome resources are the organism-centric resources, such as the Mouse Genome Informatics (MGI)  and the /zfin.org, which oThus, microarray probes should be designed on transcripts or predicted transcripts, be annotated with gene information and use the most recent transcriptome resources. Because of the exploratory nature of transcriptomics experiments, most scientists wish to detect as many different transcripts as possible, rather than to limit themselves to established transcripts and genes only. The ongoing miniaturization in microarray manufacture also allows such an approach. A simple strategy would be to design probes for all resources separately and put these together on the microarray. However, this approach causes serious difficulties in the expression analysis, such as problems in gene set enrichment and overrepresentation analysis due to redundancy of probes representing the same transcript. Here we will show a strategy to integrate the heterogeneous sequence information for transcriptome-wide microarray design and show the result of our approach for the zebrafish transcriptome.The purpose of the Microarray Design Workflow Figure  is to deThe transcript clustering is started by ordering all sequences by length and, starting with the longest sequence, mapping them onto one another using the BLAST algorithm . A similIn order to make a gene annotation for each TCs, the table of TCs is mapped to Ensembl using R-BioMart :. The TCin silico predictions  and transcript(s) to which the TC is mapped.Click here for fileZebrafish Microarray Design - Cross-hybridizing Probes. all cross-hybridizing probes are tabulated together with their sequences, the TC-id, the transcript the probe is designed on, whether the probe is designed on the sequence given by the sequence resource or is designed on the reverse complement, start of the probe on the transcript, information from the sequence resource, gene symbol, description, chromosome, strand, genomic position, other TC members, Ensembl Gene, Ensembl Transcripts and between brackets: cross-hybridizing potential to transcript, TC with the cross-hybridization bitscore. Subsequences are abbreviated by 'subs'.Click here for fileZebrafish Microarray Design - Probes designed on more than one TC. Tabulated are the probes that only could be designed to more than one TC. In the second column the TCs are given between brackets, together with the TC sequence on which the probe has been designed. Subsequences are abbreviated by 'subs'.Click here for fileZebrafish Microarray Design - Perfect hit only cross-hybridizing probes. Tabulated are the probes that have an exclusive 100% similarity to the probes they cross-hybridize with. The probes are given together with the sequences they are designed on and between brackets the TCs they cross-hybridize with plus the sequence the TC has been designed on. Subsequences are abbreviated by 'subs'.Click here for file"}
+{"text": "VMAT planning was conducted for three typical target types of prostate cancer, hypopharynx/larynx cancer and vertebral metastases, and compared to standard IMRT with respect to plan quality, number of monitor units (MU), and treatment time.VMAT was implemented on a SynergySFor prostate cancer and vertebral metastases single arc VMAT led to similar plan quality as compared to IMRT. For treatment of the hypopharynx/larynx cancer, a second arc was necessary to achieve sufficient plan quality. Treatment time was reduced in all cases to 35% to 43% as compared to IMRT. Times required for optimization and dose calculation, however, increased by a factor of 5.0 to 6.8.Similar or improved plan quality can be achieved with VMAT as compared to IMRT at reduced treatment times but increased calculation times. It has been first introduced by Otto in 2008  and implion dose ,7,9-19, ion dose . Fully iion dose -26 using\u00ae  and SynergyS\u00ae linear accelerators . VMAT optimization was performed for typical target types usually treated with IMRT at our department and compared to standard IMRT with regard to plan quality, number of monitor units, and treatment time. Patients were selected for whom treatment times required for IMRT are critical due to possible intra-fractional organ movement or patient discomfort and who therefore might benefit substantially from the advancement from IMRT to VMAT.The aim of this study was to investigate the feasibility of VMAT with the new commercial combination of Oncentra MasterPlan\u00ae linear accelerator with 6MV photons, equipped with a BeamModulator\u2122 head, an iViewGT\u2122 electronic portal imaging device, and an on-board cone-beam CT XVI is used for VMAT delivery. The BeamModulator\u2122 head has a multileaf collimator which consists of 40 leaf pairs of 4 mm width at isocenter and allows unrestricted leaf interdigitation. Fixed diaphragms limit the maximum field size of 21 cm \u00d7 16 cm, and there are no moveable jaws. Minimum and maximum number of MU per degree of gantry rotation are 0.10 MU/\u00b0 and 20.0 MU/\u00b0 respectively, minimum MU per cm leaf travel is 0.30 MU/cm, maximum gantry speed is 6.00 \u00b0/s. Maximum leaf speed is 2.4 cm/s, the dynamic minimum leaf gap 0.14 cm, and the static minimum leaf gap 0.0 cm. The maximum nominal dose rate is 500 MU/min. Seven fixed dose rate levels are available, each half the dose rate of the next higher level, continuous variation is not possible. Actual dose rates may differ from nominal dose rates by \u00b125%. During VMAT delivery the fastest combination of dose rate, gantry speed and leaf speed is automatically selected by the linac control system Precise Desktop\u00ae 7.A SynergyS\u00ae v3.3 SP1, released clinically in December 2009, on a 64 bit Windows system with 8 GB RAM and 8-core processor. For beam data modeling the above mentioned VMAT specific parameters of the linac have to be defined. Since the TPS only allows for 5 different dose rates, two dose rates had to be omitted. We kept the 5 higher and omitted the two lower dose rates, because according to the literature the main advantage of VMAT as compared to IMRT is the short treatment time, which would be prolonged if higher dose rates would be omitted. This is also in concordance with Bedford's recommendation not to use dose rates below 75 MU/min to a large extent due to instabilities of the linac below 37 MU/min [Treatment planning is performed with Oncentra MasterPlan7 MU/min . Since tFor treatment planning, beams are set up in the Beam Modeling module (BM), in which the treatment unit, energy and collimator angle are defined by the user. Gantry speed, leaf positions and dose rate are subject to optimization, the collimator angle, however, is kept constant at the predefined value for each arc. Other user defined parameters for the optimization include start gantry angle, rotation direction, arc length, gantry angle spacing between subsequent control points (2\u00b0 to 6\u00b0), maximum delivery time, number of arcs, and constrained leaf motion in cm/\u00b0.Optimization is performed in the Optimization Module, which allows the user to choose between the IMRT options \"Intensity Modulation\" (IM) with subsequent leaf sequencing, and the direct machine parameter optimization \"Direct Step and Shoot\" (DSS), and VMAT.In DSS a fluence optimization with subsequent leaf sequencing is performed for static fields for a few iterations to get an initial guess for the segments. In the next step, the gradients of the objective function are calculated with respect to leaf positions and weights, allowing direct optimization of deliverable MLC segments, which leads to improved results as compared to IM -30.VMAT optimization starts with a fluence optimization for gantry angle spacing of 24\u00b0 and subsequent MLC sequencing, generating 2 segments per gantry angle. The segments are then spread out evenly and cloned to achieve the required gantry angle spacing as defined by the user. Based on this starting point a direct machine parameter optimization is performed, taking machine restrictions into account, followed by a final accurate dose calculation and segment weight optimization. The method is a successor of and very similar to the method described in , where nContinuous delivery is discretized and approximated by the calculation of static beams separated by 2\u00b0 to 6\u00b0, depending on the user defined gantry angle spacing. The result of the accurate dose calculation is used as starting point for an automatic second optimization run to improve results . For mor\u00ae, Mosaiq\u00ae and SynergyS\u00ae for VMAT has been successfully completed with individual plan verifications within 3% dose tolerance and 3 mm distance to agreement. Validation has been performed by absolute 2D-dosimetry using the 2D-array MatriXXEvolution\u00ae . A description of the commissioning procedure and detailed results, however, is beyond the scope of this study and will be published separately.Commissioning of the system combination of Oncentra MasterPlanFor a selection of patients who had undergone or were currently under IMRT treatment at our department, VMAT plans were optimized and compared to the IMRT plans to assess plan quality achievable with VMAT. The feasibility study was performed on three patients with typical target geometries of head and neck and prostate cancer, as well as spinal cord sparing irradiation of vertebrae.3 and a boost volume of 241.7 cm3. The PTV covered the prostatic fossa and the region of seminal vesicles defined by pelvic CT with 8 mm margin for setup, organ motion and delineation uncertainties. Dose prescription was 60 Gy in 2 Gy fractions to the average of the PTV, and 70 Gy in 2 Gy fractions to the boost volume. The bladder, rectum and the femoral heads were delineated as organs at risk (OAR). The volumes of rectum and bladder, which were not overlapping with the PTV that was extended by an additional 0.8 cm margin, were used as help structures for optimization and evaluation of plan quality, referred to as \"rectum - PTV\" and \"bladder - PTV\" respectively. The feasibility study was performed for the first series only. Dose volume objectives (DVO) based on dose prescription and OAR tolerance doses are listed in table 1. A 64 year old patient with prostate cancer, pT3b, pN0, cM0, R1, with a planning target volume (PTV) of 424.1 cm3 PTV, and 452.2 cm3 boost volume. The definition of PTV and organs at risk was according to literature [2. A 52 year old male patient with cancer of the hypopharynx/larynx T4, N2c, M0, with 626.2 cmterature . Dose pr3 of the PTV and 60.7 cm3 of the GTV. The PTV was defined as the whole vertebral body with a 5 mm margin, the definition of GTV based on tumour mass identified by nuclear magnetic resonance tomography. Dose prescription was 44 Gy to the average of the PTV in fractions of 2.0 Gy and 55 Gy to the average of the GTV volume in fractions of 2.5 Gy, treated as simultaneous integrated boost (SIB). The spinal cord and the kidneys were delineated as OAR. Dose volume objectives based on dose prescription and OAR tolerance doses are listed in table 3. A 70 year old female patient with metastases in the lumbar vertebra, with a volume of 342.8 cmFor all patients the normal tissue was defined as an OAR by subtracting the PTV from the patient outline and used during optimization to prevent high dose areas outside the PTV.\u00ae, showing clear advantage for DSS [Several planning studies have been published comparing fluence modulation with subsequent leaf sequencing IM and the direct aperture optimization DSS in Oncentra MasterPlan for DSS -30. TherFor the optimization of VMAT plans, single arcs ranging from 182\u00b0 to 178\u00b0 gantry angle with a gantry angle spacing of 4\u00b0 and the leaf motion constrained to 0.5 cm/\u00b0 were used. The collimator angle was set to 45\u00b0 as suggested in , except The feasibility study showed similar plan quality at reduced delivery times and similar number of MU per fraction for VMAT as compared to IMRT in all cases:1. For the prostate case, single arc VMAT showed better dose homogeneity and target coverage, and similar, mostly even lower dose to the organs at risk. Time for optimization and dose calculation increased by a factor of 5.8, treatment time was reduced to 43%. Detailed information is given in table 2. For the case with cancer of the hypopharynx/larynx, single arc VMAT showed similar target coverage and better sparing of the parotids, but deteriorated homogeneity as compared to IMRT. Better overall plan quality including target coverage, homogeneity inside the PTV, as well as OAR sparing could be achieved with dual arc VMAT. Even the relative volume of the normal tissue, receiving doses between 20.0 Gy and 50.0 Gy is smaller in case of the dual arc treatment. Only the relative volume of the normal tissue receiving between 5.0 Gy and 20.0 Gy is slightly larger. Detailed information is given in table 95% for the PTV higher, doses to the kidney were also higher but still below the tolerance and fulfilling the DVO used in optimization. Time for optimization and dose calculation increased by a factor of 5.0, treatment time was reduced to 41%. Since patients with bone metastases suffer from pain and are not able to keep the position for a long time, the VMAT plan was considered superior because of the reduced treatment time. Detailed information is given in table 3. For the patient with metastases in the lumbar vertebra, single arc VMAT showed similar plan quality as compared to IMRT. Doses to the GTV were similar, median dose and DPatient 1 and 3 have actually been treated with VMAT after successful completion of commissioning, patient 2 had already finished treatment.\u00ae v.3.3 allows creating VMAT plans with similar or better plan quality as compared to IMRT which can be delivered in substantially reduced treatment time on an Elekta SynergyS\u00ae linear accelerator. For the treatment of prostate cancer and vertebral metastases, the required plan quality could be achieved with one single arc VMAT, which is in agreement with the results published for other types of equipment [\u00ae  could not be confirmed. In this case, however, plan comparison was performed for simultaneous treatment of three target levels, which requires certain dose heterogeneity inside the target.The VMAT optimization tool implemented in Oncentra MasterPlanquipment ,18,24,35quipment ,24,36. Tquipment , who repThe applicability of the system to simultaneous integrated boost concepts has been demonstrated for the treatment of vertebral metastases. In this case, a single arc was sufficient to achieve the required plan quality. The same concept can be applied for SIB treatments of other target types like prostate or head and neck cancers. It might even be possible that single arc treatments are in general suitable for SIB concepts due to the required dose heterogeneity inside the target, which would also explain the results of Bertelsen  mentione\u00ae v3.3, segment shapes and weights are subject to optimization, which is one of the main differences to the treatment planning system ERGO++\u00ae: In ERGO++\u00ae segment shapes have to be defined by the user prior to optimization, and only the segment weights are optimized.In the VMAT solution implemented in Oncentra MasterPlan\u00ae is therefore highly dependent on the individual user's experience in creating suitable segment shapes. The VMAT solution implemented in Oncentra MasterPlan\u00ae v3.3 in contrary does not require any user input for the segment shapes. Segment shapes and weights are resulting from the optimization process and the resulting plan quality is therefore less dependent on the individual user.The quality of the VMAT plans resulting from optimization in ERGO++The number of monitor units per fraction in this study was similar for VMAT and IMRT, a significant reduction as reported for Varian linacs could not be observed , since tThe combination of plan quality and treatment time shows clear advantage of VMAT over IMRT: Treatment time is a crucial factor especially for patients who suffer from pain or are not able to keep a certain position for a longer time, as it is the case e.g. for patients with bone metastases, or for patients with significant internal organ movement, e.g. patients with prostate cancer, for which the actual delivered dose distribution might differ significantly from the planned dose distribution due to intra-fractional movement. In these cases even a single arc leads to the required plan quality, allowing reducing overall treatment time from 11 minutes to well below 5 minutes. For the patient with hypopharynx/larynx cancer the dual arc VMAT showed better plan quality at only 33% of the treatment time, which reduces patient discomfort in the rigid mask system. The reduction in delivery time leads to better patient comfort and possibly also quality of delivery, and simultaneously reduces the workload and increases availability of the linac.\u00ae.The only drawback found for VMAT as compared to IMRT was the increased calculation time. This, however, has no impact on patient treatment or on the workload but is only affecting availability of the treatment planning station. Workload for the planner is virtually the same for VMAT as for IMRT, since the steps of the planning procedure, which require user interaction, like definition of structures, beam setup, definition of DVO, are the same in both cases. In the future calculation times may be reduced using a processor with more than 8 cores or performing the dose calculation on the GPU processor, as it will be implemented in the next version of Oncentra MasterPlan\u00ae has the potential to produce better plan quality requiring less delivery time as compared to IMRT. However, dedicated planning studies should be performed, varying the user definable parameters e.g. maximum treatment time, number of arcs, and gantry angle range, to identify the best parameter set to achieve optimal combination of plan quality and treatment time for each target type.It could be shown that VMAT planning with Oncentra MasterPlan\u00ae allows achieving comparable or superior plan quality with VMAT as compared to IMRT. Times required for optimization and dose calculation are increased, the number of monitor units per fraction is similar, and treatment times are strongly reduced.Oncentra MasterPlanCT: Computed Tomography; DSS: Direct Step and Shoot optimization; DVH: Dose Volume Histogram; DVO: Dose Volume Objective; GTV: Gross Tumour Volume; IM: Intensity Modulation with subsequent sequencing; IMRT: Intensity Modulated Radiation Therapy; MLC: Multi-Leaf Collimator; MU: Monitor Units; OAR: Organ at Risk; PTV: Planning Target Volume; SIB: Simultaneous Integrated Boost; TPS: Treatment Planning System; VMAT: Volumetric Modulated Radiation TherapyThis work was partly supported by Theranostic.BD conceived of and designed the study, performed treatment planning and plan comparison and drafted the manuscript. KW performed part of the treatment planning. OK helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "Poultry production has been widely criticized for its negative environmental impact related to the quantity of manure produced and to its nitrogen and phosphorus content. In this study, we investigated which traits related to excretion could be used to select chickens for lower environmental pollution.The genetic parameters of several excretion traits were estimated on 630 chickens originating from 2 chicken lines divergently selected on apparent metabolisable energy corrected for zero nitrogen (AMEn) at constant body weight. The quantity of excreta relative to feed consumption (CDUDM), the nitrogen and phosphorus excreted, the nitrogen to phosphorus ratio and the water content of excreta were measured, and the consequences of such selection on performance and gastro-intestinal tract (GIT) characteristics estimated. The genetic correlations between excretion, GIT and performance traits were established.Heritability estimates were high for CDUDM and the nitrogen excretion rate . The other excretion measurements showed low to moderate heritability estimates, ranging from 0.10 for excreta water content to 0.22 for the phosphorus excretion rate. Except for the excreta water content, the CDUDM was highly correlated with the excretion traits, ranging from -0.64 to -1.00. The genetic correlations between AMEn or CDUDM and the GIT characteristics were very similar and showed that a decrease in chicken excretion involves an increase in weight of the upper part of the GIT, and a decrease in the weight of the small intestine.In order to limit the environmental impact of chicken production, AMEn and CDUDM seem to be more suitable criteria to include in selection schemes than feed efficiency traits. In terms of quantity, FEW and DEW were 70.2% and 118.3% higher in D- birds, respectively. The D+ birds also excreted 35.1% less water than D- birds. This difference partly reflected a difference in feed consumption, which was 27.2% higher in D- birds between 17 and 23d. However, even when correcting for this difference in feed consumption, FEW, DEW, and the gross quantity of water were still 36.8, 67.9, and 25.1% higher in D- birds. Furthermore, the D+ birds excreted 49.0 and 60.6% less fresh and dry excreta than D- birds for the same BW at 23 d of age. In terms of the composition of excreta, the relative nitrogen and phosphorus excretion levels were 34.9 and 19.0% lower for D+ than for D- birds, respectively. As the difference between lines was greater for nitrogen than for phosphorus, the nitrogen to phosphorus ratio in excreta was 20.3% higher for D+ than for D- birds. .Heritability estimates of BW23, WG, AMEn, FCR, RFI, FI and the gastro-intestinal tract can be found in de Verdal et al. . The herg = -0.05) whereas the latter was very highly correlated with this trait (rg = -0.87). The genetic correlation between FEW/BW or DEW/BW and FEW/FI, NE/NI, PE/PI and NE/PE were high (ranging from 0.36 to 0.82). Excretion of nitrogen and phosphorus was highly correlated (rg = 0.74). Finally, it should be noted that the balance between N and P in excreta was mainly correlated with N excretion (rg = 0.58), but very poorly with P excretion (rg = -0.11) but standard errors of the parameters could not be estimated.The genetic correlations between the various excretion traits are shown in Table The genetic correlations between excretion traits and performance traits are shown in Table The expected response on excretion traits to direct selection or to indirect selection of AMEn, CDUDM, FCR and RFI are shown in Table g = 0.47) and with the density of the 3 intestinal segments (from 0.42 to 0.72). An increased water excretion rate was genetically linked to a shorter and denser intestine , and with a lighter proventriculus (rg = -0.48).The genetic correlations between the excretion traits and GIT characteristics are shown in Table g between 0.63 and 0.76), but only moderate correlations with densities (rg = 0.39 on average). In the same way, FEW/BW and DEW/BW were positively correlated with intestine relative weight and density, the only non significant correlation being found between DEW/BW and DD. Phosphorus excretion was also moderately correlated with PRW, and nitrogen excretion with duodenum weight relative to BW23. The similarity of genetic correlations of anatomic traits with NE/NI and PE/PI ratios mean that the NE/PE ratio was weakly or moderately correlated with anatomic characteristics. Indeed, the NE/PE ratio was only moderately correlated with proventriculus relative weight and with jejunum relative weight and length.A high positive genetic correlation was observed between phosphorus and nitrogen excretion and relative weights of jejunum and ileum , which makes convergence more difficult. It was for example the case when FEW/BW with low h\u00b2 was included simultaneously with DEW/BW, with which it was very highly correlated. Similarly, even if FCR and AMEn had already been shown to be strongly correlated (-0.70 ), the geExcretion traits were moderately heritable, showing that it should be possible to include such traits in poultry selection. The estimated heritability of PE was much higher than that reported by Zhang et al.  and AnkrIn the present study, we found that D+ birds had a 28.2% greater CDUDM than D- birds, showing that digestive utilization was improved in D+ compared to D- birds. This could be explained by the genetic correlations between CDUDM and GIT morphology. Indeed, it seems that selection on high CDUDM would increase the relative weight of the upper part of the GIT (proventriculus and gizzard) and conversely decrease the relative weight and the density of the small intestine, consistent with previous results . A largeThis higher digestive utilisation in D+ birds leads to a 41.3 and 54.1% reduction in FEW and DEW, respectively, compared to D-. These differences are also present at later ages. Furthermore, the commercial line used at the beginning of the selection experiment excreted 31.5% more DEW/FI between 21 and 53 d of age than D+ birds . SelectiHowever, most of the studies related to environmental problems due to the spreading of manure focus on N and P content  and theiFurthermore, NE/NI and PE/PI ratios were more genetically correlated with jejunum and ileum relative weights and densities than with those of the duodenum. This illustrates the major contribution of the lower part of the intestine in N and P absorption ,33. Neve2O5) that can be spread on fields to 170 kg. ha-1 and 100 kg.ha-1, respectively, the ideal ratio of N to P2O5 on spread manure should thus be 1.7 [2O5 that would be found in the manure would be 1.95 and 2.33 for the D+ and the D- birds, respectively. However, since 50% of the N excreted by chickens is lost between excretion and spreading [2O5 compared to N. This suggests first that N losses should be limited to increase the N/P2O5 ratio in manure and secondly that this limitation should take into account the genotype of birds. Indeed, N losses in manure should be limited to 15% in D+ birds and 37% in D-birds, whereas the usual value is closer to 50%.These high levels of differences in N and P excretion between D+ and D- birds could explain why the ratio of the NE to PE was 25.4% higher in D- than in D+ birds. French and European regulations limit the amounts of N and phosphates  allow measuring these traits at a very moderate cost.Besides AMEn or CDUDM selection, direct selection on excretion traits could be considered. Using equation , it appeOur genetic results indicate that limiting the environmental impact of chicken production by selection could be achieved by selecting on AMEn as well as on the CDUDM. According to the estimated genetic correlations, a decrease in chicken excretion is associated with an increase in proventriculus and gizzard relative weights, which would be likely to improve nutrient accessibility in the small intestine and thus the digestibility. Because of the increased competition between humans and animals for access to food  and the use of non-renewable materials (such as inorganic P) in animal nutrition, the adaptation of birds to alternative diets of lower nutritional quality will become an important issue. This study highlights that there is wide genetic variability, and this may be used to improve feed digestibility and thus limit the excretion responsible for environmental pollution. Finally, even if classical selection criteria as FCR would reduce environmental impact of poultry production, greater responses could be expected from selection on digestive efficiency.The authors declare that they have no competing interestsHDV, AN, ELB and SMG contributed to the experimental design, data analysis, interpretation of data and manuscript preparation. HC contributed to the data analysis. DB contributed to the use of NIRS in digestibility measurements. NM and SU assisted in the acquisition of data. All authors read and approved the final manuscript."}
+{"text": "N\u00a0=\u00a0122) watched analogue traumatic and neutral picture stories. They were then randomly allocated to 20\u00a0min of either imaginal exposure, autobiographical memory elaboration, or a control condition designed to prevent further processing of the picture stories. A blurred picture identification task showed that neutral objects that preceded traumatic pictures in the stories were subsequently more readily identified than those that had preceded neutral stories, indicating enhanced priming. There was also an evaluative conditioning effect in that participants disliked neutral objects that had preceded traumatic pictures more. Autobiographical memory elaboration reduced the enhanced priming effect. Both interventions reduced the evaluative conditioning effect. Imaginal exposure and autobiographical memory elaboration both reduced the frequency of subsequent unwanted memories of the picture stories.Unwanted memories of traumatic events are a core symptom of post-traumatic stress disorder. A range of interventions including imaginal exposure and elaboration of the trauma memory in its autobiographical context are effective in reducing such unwanted memories. This study explored whether priming for stimuli that occur in the context of trauma and evaluative conditioning may play a role in the therapeutic effects of these procedures. Healthy volunteers ( \u25ba We investigated cognitive mechanisms of interventions that reduce reexperiencing after trauma. \u25ba Neutral objects shown in traumatic picture stories were later preferentially identified and evaluated negatively. \u25ba Memory elaboration balanced perceptual priming for objects from traumatic and neutral stories. \u25ba Imaginal exposure and autobiographical memory elaboration led to more balanced emotional evaluations. \u25ba Interventions may prevent selective perceptual or emotional responses to trauma reminders. A rangestrong perceptual priming for stimuli that occurred shortly before and during the traumatic event.This study focuses on two memory processes that may be involved in the triggering of unwanted trauma memories: perceptual priming and evaluative conditioning. Interest in perceptual priming was generated by clinical observations suggesting that a particularly wide range of stimuli can trigger unwanted trauma memories e.g., . InterviSome studies have tested the hypothesis that stimuli that are associated with the trauma are more strongly primed in people with PTSD than in those without PTSD. Participants encoded trauma-related and control stimuli (mainly words or sentences) and priming was tested later with word-stem completion or perceptual identification tasks. The results mostly support the hypothesis of greater perceptual priming for material associated with the trauma in people with PTSD compared those without PTSD . These eExperimental analogue studies provided initial support for the role of perceptual priming in the development of analogue intrusive trauma memories in healthy controls . For exaOther memory processes of interest in the triggering of unwanted trauma memories are learned associations e.g., . It has The present experimental analogue study used Hypothesis 1 : On the basis of studies by Hypothesis 2 (Effects of elaboration on enhanced priming): On the basis Hypothesis 3 . On the basis of studies on evaluative conditioning . On the basis of research showing that evaluative conditioning can be modified by subsequent intervention : On the basis of the efficacy of prolonged exposure \u00a0=\u00a00.34, p\u00a0=\u00a00.844, age, F \u00a0=\u00a00.98, p\u00a0=\u00a00.378, trait anxiety, F \u00a0=\u00a01.50, p\u00a0=\u00a00.228 or state anxiety, F \u00a0=\u00a01.72, p\u00a0=\u00a00.182.Participants were 122 volunteers who were recruited by advertisements in the local area in south London and via emails to staff and students of King\u2019s College London. They were reimbursed with \u00a315 for their time and travel expenses. Volunteers were excluded if they reported a history of trauma, current blood/injury phobia or severe depression. 3preceding objects). The second picture (presented for 20\u00a0s) depicted the plot of the story and showed something traumatic or neutral happening. It contained one central object that was important for the course of the story, and determined whether the content was traumatic or neutral. The third and last picture (presented for 15\u00a0s) showed the final outcome for the main character of the story. It focused on and underlined the traumatic versus neutral content of the story. The unpleasant and neutral picture stories were matched for the number of males, females, and objects occurring in them, and whether the event happened indoors or outdoors. For example, one unpleasant story contained a dog killing a man and the matching story depicted a cat stealing a sandwich from its owner. A list of the content of all stories is presented in the\u00a0The experimental software for all parts of the experiment was programmed with SuperLab. Picture stories and memory tests were presented on a 15\u2019\u2019 screen of an Apple Macintosh computer. The picture stories were the same as in Participants were told that it was the purpose of the experiment to test how pictures affect people\u2019s emotions. They were asked to watch the pictures closely and to imagine that they were present at the scene. After each picture story, they were asked to rate the pictures for pleasantness and arousal. There was no indication that memory for the pictures would be tested later.Picture stories were presented in two blocks of \u201ctraumatic\u201d and neutral stories, in counterbalanced order. Blockwise presentation was chosen to prevent crossover of negative emotions produced by unpleasant picture stories to neutral ones. Order of presentation did not influence the results. Between blocks participants had a 5-min break. The order of the stories within each block was randomized and different for every participant.3.1Participants were randomly allocated to one of three experimental conditions that followed the picture story task.3.1.1M\u00a0=\u00a07.20, SD\u00a0=\u00a01.75.Instructions were modeled on imaginal exposure . Partici3.1.2This group received the same instructions as in 3.1.3readilly, examined, assuming). The fourth task comprised groups of words, and participants had to find the word that was closest in meaning to a word typed above the group . No task contained trauma-related words.Participants in the control group were told that the experiment dealt not only with the processing of pictures but also with the processing of words. They were given several sheets that each contained a different word task, and were told that they should try to complete as many of these tasks as possible in the next 20\u00a0min. The first task contained a list of neutral words and participants had to explain the meaning of these words (e.g. \u2018connect\u2019 means \u2018join together\u2019). The second task contained phrases, and participants had to identify words that were not correctly spelled given the context of the phrase (\u2018a honey be\u2019). The third task was to identify which word of a group of four was misspelled  of objects were created for each task. Each set contained one of the preceding objects from each story and, in the recognition task, half of the central objects from unpleasant and neutral scenes. Half of the participants saw objects from set 1 in the perceptual priming task and objects from set 2 in the recognition task. For the other half of the participants, sets were reversed. Picture set did not influence the results.3.2.1n\u00a0=\u00a024) were unprimed objects that had not featured in the picture stories . These unprimed objects were matched for size to the primed objects from the picture stories.A blurred object identification task assessed priming as visual perceptual priming leads to an enhanced identification rate for previously seen objects. In order to decrease the chance that participants noticed that the task was a memory test, only the preceding objects from the stories  were included. Furthermore, the majority of items  should be identified at baseline rates, resulting in a mean incremental identification probability of 0.3.2.2M\u00a0=\u00a045.20, SD\u00a0=\u00a012.77, objects from neutral stories, M\u00a0=\u00a044.56, SD\u00a0=\u00a012.32; distractor objects, M\u00a0=\u00a044.37, SD\u00a0=\u00a011.23).To assess evaluative conditioning, participants rated how much they liked each picture of the blurred picture task on a scale from 0 \u2018not at all\u2019 to 100 \u2018very much\u2019, with 50 representing \u2018neutral.\u2019 3.2.3The recognition task tested explicit memory performance for objects from the picture stories. It included both central and preceding objects from the picture stories. For each \u201cold\u201d object from the picture stories, a parallel new object was chosen that looked somewhat different in appearance. These parallel new objects matched the objects from the picture stories in size and object type . Objects were presented on a computer screen in successive, random order which was different for each participant. Participants were asked to indicate whether or not they had seen the object previously in the stories by pressing the corresponding keys on the computer keyboard.3.3The Past Experience Questionnaire screens participants for a trauma history, blood/injury phobia, and severe depression. Participants who met any of these criteria were excluded from the study. Participants also completed the state and trait versions of the State-Trait Anxiety Inventory  and a 4-3.4At 4 weeks and 3 months, participants were interviewed on the telephone. They were asked about any unwanted memories of the picture stories that had occurred without an apparent reason. They rated the frequency of such memories in the past 2 weeks on a scale from 0 \u2018never\u2019 to 7 \u2018more often than once a day\u2019.3.5The study was approved by the local Ethics Committee. Participants received an information sheet about the study and were given further information on the telephone when arranging the appointment. They were informed in writing and by the experimenter that the study involved watching some unpleasant pictures and that they could withdraw at any time without having to give a reason. On arrival at the laboratory, participants gave written consent. They then completed the Past Experience Questionnaire, the STAI (state version) and the first Mood Rating. Participants were then given oral and written instructions for watching the picture stories, and watched the two blocks of picture stories. They completed a Mood Rating after each of the blocks. The experimental manipulation (20\u00a0min) followed. Participants completed another Mood Rating and then had a 10-min break, during which the experimenter served a drink and conversed with them about unrelated matters. They then completed the perceptual priming task and the emotional evaluation ratings, followed by completion of the STAI (trait version) and the object recognition task.The experimenter made sure that participants felt well before leaving and gave participants her contact details, encouraging them to get in touch if they felt in any way distressed about the experiment. However, none of the participants took up this offer and none reported that they found the experiment too distressing.Participants completed the Intrusive Memories Interview over the telephone at 4 weeks and 3 months after the experiment. At 3 months they were debriefed about the purposes of the study.3.63.6.1The priming index was computed to compare priming for objects from \u201ctraumatic\u201d versus neutral stories. The priming index was the difference between the incremental identification rates for primed minus unprimed objects. Greater scores indicate greater priming.3.6.2A parallel evaluative conditioning index was calculated as the difference between the emotional evaluations for the primed minus the unprimed objects. Negative scores indicate greater conditioned emotional evaluations.3.6.3d\u2019) and response bias (c) scores were calculated, for both preceding and central objects. Sensitivity is a standard measure of recognition memory performance that measures how well participants discriminated between objects from the stories and parallel objects that they had not seen before, calculated as d\u2019\u00a0=\u00a0probit (hits)\u00a0\u2212\u00a0probit . Response bias is a measure of leniency in endorsing an object as \u201cold\u201d, calculated as c\u00a0=\u00a0\u22120.5*(probit (hits)\u00a0+\u00a0probit ).Data analysis followed signal detection theory (SDT) . From th3.6.4Data were analyzed with the General Linear Models (GLM) and\u00a0CROSSTABS procedures in SPSS 15.0. Planned contrasts were used to test the hypotheses. Greenhouse\u2013Geisser corrections were used if appropriate. We report one-tailed significance levels for planned contrasts as the hypotheses were uni-directional, and two-tailed significance levels for other comparisons. Details of the analyses are found below.44.1In no case did participants falsely identify an object from the picture stories when another object was presented.4.1.1M\u00a0=\u00a00.02, SD\u00a0=\u00a00.14 versus M\u00a0=\u00a0\u22120.03, SD\u00a0=\u00a00.18, F \u00a0=\u00a011.88, p\u00a0<\u00a00.001, \u03b72\u00a0=\u00a00.089.To test whether watching the picture stories led to perceptual priming, incremental identification rates for the objects from picture stories (primed objects) were compared with those of matched distracter items that participants had not seen before (unprimed objects). Primed objects from picture stories were identified with greater probability than unprimed objects without prior exposure. 4.1.2F\u00a0=\u00a09.29, p\u00a0=\u00a00.003, \u03b72\u00a0=\u00a00.071. Consistent with the enhanced priming hypothesis, blurred pictures of neutral objects that had preceded the \u201ctraumatic\u201d pictures in the picture stories were more readily identified, M\u00a0=\u00a00.12, SD\u00a0=\u00a00.19, than those that preceded neutral pictures, M\u00a0=\u00a00.05, SD\u00a0=\u00a00.19.The results for the perceptual priming task (priming index) are presented in 4.1.3F \u00a0=\u00a03.03, p\u00a0=\u00a00.042, \u03b72\u00a0=\u00a00.025. Enhanced priming for objects from \u201ctraumatic\u201d stories was evident in both the control, F\u00a0=\u00a05.15, p\u00a0=\u00a00.029, \u03b72\u00a0=\u00a00.114, and the imaginal exposure conditions, F\u00a0=\u00a06.93, p\u00a0=\u00a00.012, \u03b72\u00a0=\u00a00.148, whereas the autobiographical memory elaboration group showed equal priming for objects from \u201ctraumatic\u201d and neutral picture stories, F\u00a0=\u00a00.11, p\u00a0=\u00a00.740, \u03b72\u00a0=\u00a00.003.The planned contrast comparing the enhanced priming effect  for the autobiographical memory elaboration group with the other two groups was significant, 4.1.4F\u00a0=\u00a08.44, p\u00a0=\u00a00.004, \u03b72\u00a0=\u00a00.065. As expected, neutral objects that had preceded \u201ctraumatic\u201d pictures were evaluated more negatively, M\u00a0=\u00a0\u22123.30, SD\u00a0=\u00a07.00, than those that preceded neutral pictures, M\u00a0=\u00a0\u22121.18, SD\u00a0=\u00a06.58.The results of the emotional evaluations of the objects from the picture stories are presented in 4.1.5F \u00a0=\u00a03.21, p\u00a0=\u00a00.038, \u03b72\u00a0=\u00a00.026. Participants in the control group showed the evaluative conditioning effect and gave more negative emotional evaluations for objects from traumatic stories, F\u00a0=\u00a013.85, p\u00a0=\u00a00.001, \u03b72\u00a0=\u00a00.257. This was not the case for the two intervention groups, imaginal exposure, F\u00a0=\u00a00.30, p\u00a0=\u00a00.587, \u03b72\u00a0=\u00a00.007; autobiographical memory elaboration group, F\u00a0=\u00a01.39, p\u00a0=\u00a00.245, \u03b72\u00a0=\u00a00.034.The planned contrast comparing the conditioned evaluation effect  for the two intervention groups and the control group was significant, 4.2F \u00a0=\u00a03.46, p\u00a0=\u00a00.032, \u03b72\u00a0=\u00a00.029. The imaginal exposure, M\u00a0=\u00a00.46, SD\u00a0=\u00a00.87, and autobiographical memory elaboration groups, M\u00a0=\u00a00.56, SD\u00a0=\u00a01.27, reported fewer unwanted memories than the control group, M\u00a0=\u00a01.02, SD\u00a0=\u00a01.93. At 3 months, very few participants reported unwanted memories, but the intervention groups were still less likely to report unwanted memories, 14.3% of the control group and 5.2% of the intervention groups, \u03c72 \u00a0=\u00a02.69, p\u00a0=\u00a00.05.The planned contrast testing Hypothesis 5 (intervention effect on unwanted memories) showed the expected difference between the intervention groups and the control group in the frequency of intrusive memories at 4 weeks, 4.34.3.1p\u00a0>\u00a00.41, nor interactions, all p\u00a0>\u00a00.14, with experimental group. As to be expected on the basis of eye-witness research \u00a0=\u00a0176.63, p\u00a0<\u00a00.001, \u03b72\u00a0=\u00a00.600. In contrast to the perceptual priming task, there was no main effect of story context  in the sensitivity with which the objects were identified in the recognition task, F\u00a0=\u00a00.91, p\u00a0=\u00a00.341, \u03b72\u00a0=\u00a00.008, There was an interaction between story context and object importance, F \u00a0=\u00a06.48, p\u00a0=\u00a00.012, \u03b72\u00a0=\u00a00.052. Post-hoc analyses showed that this interaction was due to somewhat better discrimination of central objects from neutral compared to central objects from \u201ctraumatic\u201d stories, F \u00a0=\u00a04.21, p\u00a0=\u00a00.042, \u03b72\u00a0=\u00a00.034. There was no difference in the sensitivity of discriminating preceding objects from traumatic and neutral stories, p\u00a0>\u00a00.18.The results of the object recognition task are presented in research , there wp\u00a0>\u00a00.69, nor interactions, all p\u00a0>\u00a00.16, with experimental group. There was a main effect of story context, F \u00a0=\u00a09.01, p\u00a0=\u00a00.003, \u03b72\u00a0=\u00a00.071, which was qualified by a trend for an interaction between story context and object importance, F \u00a0=\u00a02.81, p\u00a0=\u00a00.096, \u03b72\u00a0=\u00a00.023. Further analyses showed that participants used a more liberal response criterion for central objects from \u201ctraumatic\u201d stories than for central objects from neutral stories, F \u00a0=\u00a08.04, p\u00a0=\u00a00.005, \u03b72\u00a0=\u00a00.063. However, there was no effect of story context for the preceding objects, F \u00a0=\u00a01.94, p\u00a0=\u00a00.167, \u03b72\u00a0=\u00a00.016.r\u00a0=\u00a00.10, p\u00a0=\u00a00.289, and, r\u00a0=\u00a00.08, p\u00a0=\u00a00.387, respectively.Sensitivity and response bias for preceding objects from \u201ctraumatic\u201d stories in the recognition task did not correlate with identification rates in the perceptual priming test, 4.3.2F \u00a0=\u00a034.13, p\u00a0<\u00a00.001, \u03b72\u00a0=\u00a00.225, and a group\u00a0\u00d7\u00a0phase interaction F \u00a0=\u00a03.66, p\u00a0<\u00a00.003, \u03b72\u00a0=\u00a00.058. Separate group comparisons for each experimental phase showed a significant group difference in negative mood after the experimental manipulation, F \u00a0=\u00a04.77, p\u00a0=\u00a00.010, \u03b72\u00a0=\u00a00.075, but not for any of the other time points, all ps\u00a0>\u00a0.23. The imaginal exposure group reported more negative mood after the experimental manipulation than the control group, p\u00a0=\u00a00.003. The memory elaboration group did not differ significantly from the other two groups.A 3\u00a0\u00d7\u00a04 GLM with experimental group as the between subject factor and experimental phase  as the within-subject factor showed significant effects of experimental phase, 5This study was motivated by two clinical observations. First, intrusive trauma memories often appear to be triggered by cues that are perceptually similar to the intrusions, or to stimuli that immediately preceded the respective sensation during the trauma e.g., . Second,The two memory mechanism under investigation showed the expected pattern of differences between objects that were perceived in the context of trauma and those that occurred in neutral contexts. In line with Hypothesis 1 (enhanced priming effect), the study replicated previous results  that neuThere has been a debate in the literature on the influence of explicit memory on the performance in implicit memory tasks such as priming tests e.g., . The preevaluative conditioning effect), participants reported that they liked neutral objects that had preceded the \u201ctraumatic\u201d pictures less than those that had preceded neutral pictures. These results are in line with the general literature on evaluative conditioning , both interventions decreased the likelihood of subsequent intrusive memories. This result is in line with the effects of imaginal exposure  and cognThe effects of the interventions on the two memory mechanisms under investigation differed, as expected. In line with Hypothesis 2 (effects of elaboration on enhanced priming), a post-\u201ctrauma\u201d intervention designed to increase the elaboration of the picture stories in the context of the participants\u2019 other autobiographical experiences eliminated the enhanced priming effect. This result replicates both sets of stories. The findings parallel research into learned fear responses showing that learned associations are not erased by subsequent extinction training, but can be inhibited , both interventions affected emotional evaluations of the stimulus material from the picture stories. In contrast to the control group who reported to dislike the stimuli from the \u201ctraumatic\u201d picture stories more than those from the neutral stories, and thus showed the expected conditioned evaluation effect, participants who had received the interventions gave similar ratings regardless of the context in which they had experienced the objects. This demonstrates that conditioned evaluations can be modified in several ways. The lack of differential ratings to objects from the trauma stories may reduce the probability that negative mood is triggered by similar cues in the environment, which may then trigger trauma memories. It is of interest to note that the intervention groups did not give neutral ratings. Rather, they reported that they disliked objects from nhibited . Both thProlonged Exposure treatment protocol (Cognitive Therapy for PTSD (The interventions used in this experiment delivered components of complex treatment protocols without the context of a therapeutic relationship to study their pure effects. The pattern of results may be therefore be different for the full treatment programs. For example, the imaginal exposure condition used in this study did not affect priming. This procedure used the visualization component of the protocol  and partprotocol . Similarfor PTSD , nor othIt is noteworthy the term memory elaboration as used in this study differs from the way it is interpreted by The study had several limitations. First, the experiments used an analogue design and it remains unclear to what extent the results would generalize to traumatic events that would meet DSM-IV  criteria"}
+{"text": "Mycobacterium tuberculosis and methicillin-resistant Streptococcus aureus, the solid-state nanopore diagnostic platform may be used to detect large insertions or deletions, small insertions or deletions, and even single-nucleotide variations in bacterial DNA. We further show that Bayesian classification of test samples can provide highly confident pathogen typing results based on only a few tens of independent single-molecule events, making this method extremely sensitive and statistically robust.In clinical settings, rapid and accurate characterization of pathogens is essential for effective treatment of patients; however, subtle genetic changes in pathogens which elude traditional phenotypic typing may confer dangerous pathogenic properties such as toxicity, antibiotic resistance, or virulence. Existing options for molecular typing techniques characterize the critical genomic changes that distinguish harmful and benign strains, yet the well-established approaches, in particular those that rely on electrophoretic separation of nucleic acid fragments on a gel, have room for only incremental future improvements in speed, cost, and complexity. Solid-state nanopores are an emerging class of single-molecule sensors that can electrophoretically characterize charged biopolymers, and which offer significant advantages in terms of sample and reagent requirements, readout speed, parallelization, and automation. We present here the first application of nanopores for single-molecule molecular typing using length based \u201cfingerprints\u201d of critical sites in bacterial genomes. This technique is highly adaptable for detection of different types of genetic variation; as we illustrate using prototypical examples including  Furthermore, the complexity and quantity of sequencing data far exceeds the minimum information required to efficiently and accurately diagnose a patient. For example, bioinformatics studies suggest that a panel of just 30\u201350 single nucleotide variations (SNVs) could be used to uniquely identify thousands of strains of Mycobacterium tuberculosis [Subtle genetic changes in bacteria can produce large variations in factors affecting pathogenicity, such as toxicity, antibiotic resistance, and virulence. These genetic variations are not only used to trace the epidemic and phylogenetic relationships among strains of bacteria, but are also critically important in clinical settings for proper patient diagnosis and treatment. Most existing approaches require sample incubation and growth over the course of multiple days prior to testing, and nearly all require expert handling of samples and interpretation of results. Traditional phenotypic typing techniques such as serotypes, biotypes, phage-types, and antibiograms lack the necessary sensitivity to distinguish between closely related pathogen strains, and therefore fail to adequately capture these critical variations for clinical applications. Gel-based techniques such as restriction fragment length polymorphism (RFLP) or cleaved amplified polymorphic sequences (CAPS) require a large amount of time and results are not easily compared or transferred among labs. Next-generation sequencing is an increasingly popular method of fully characterizing bacterial strains  and may rculosis . Yet SNVx membrane [Solid-state nanopores may be used to discriminate the lengths of unlabeled individual biopolymers such as DNA molecules across a wide range of lengths , 6. Biopmembrane , 8. WhenMycobacterium tuberculosis (virulent vs. avirulent) and Streptococcus aureus (methicillin-resistant vs. multi-drug resistant). This highly versatile combination of rapid length and digest discrimination, spanning several orders of magnitude of possible genomic variation size, in a single, parallelizable device, could be extended to probe a large panel of critical sites within a genome for point-of-care determination of critical pathogenic properties and sequence typing.Here we describe and practice a novel detection scheme  for moleIB) and the dwell time of each molecule within the pore (tD). Both parameters have been shown to grow nonlinearly with DNA length, forming the basis for fragment length separation in the nanopore system. The statistical distributions of these independently measured quantities may be used to distinguish between analytes of different lengths, such as DNAs [tD) in solid-state nanopores measured for different DNA lengths (l), are empirically described by a power law: tD \u223c l\u03b1 where \u03b1 = 1.38\u00b10.02, which has been reproduced by multiple experimental approaches [tD, note that the difference in log(tD) for two sequences (lengths l0 and l0 + \u0394l) is more apparent for shorter length l0 as compared with the insertions and deletions \u0394l (i.e. when \u0394l/l0 \u223c 1) according to The simplest form of nanopore translocation analysis involves the measurement of the depth of each current blockade  is required, the measured histograms of either \u0394IB or tD may overlap significantly, making discrimination between these molecules difficult. Combinations of multiple fragment lengths within a sample pose additional challenges, as their more complicated distributions may overlap or otherwise preclude simple contour cluster separation.If the presence of two fragment lengths must be identified from within a single sample, it is desirable that their distributions of \u0394l are set by: 1) sample preparation parameters and limitations; for example, robust and fast PCR amplification is most easily achieved for fragment lengths of ~102\u2013103 bp [l0 are set by nanopore sensitivity; while several groups have demonstrated detection of small DNA fragments (<50 bp) [l0 on the order of ~100 bp is more reliable since it is readily detectable in small nanopores with no additional modifications [l0, ranging up to a few thousand base pairs maximum length for l0 + \u0394l. Many types of common genetic variations used for strain typing fall within this size range. For example, one complete IS6110 (insertion-like sequence element) insertion in M. tuberculosis is 1358 bp [S. aureus (MRSA) have many insertions and deletions in the range 47 bp\u2014643 bp that affect their pathogenicity [l, we turn to the exquisite sequence specificity of digestion by restriction enzymes, which can identify sequence polymorphisms down to a single nucleotide variation.In the context of sequence typing, identification of fragments by sizing will indicate the presence of specific insertions and deletions that may enhance or reduce pathogenicity or otherwise uniquely identify a pathogenic strain. Upper bounds on \u03942\u2013103 bp  and 2) n<50 bp)  we find 0 bp  we  1358 bp . At the genicity . To deteUsing these design principles, we present here two alternative modes of detection that illustrate the wide range of genomic variations that may be detected using a single sensor. For large insertions or deletions (IB) and dwell time (tD). From comparison of l several times larger than the base length (here: \u0394l:l0 = 9:1) are indeed easily distinguishable  differentiate l0  = 0.415 for l0 = 100 bp, and \u0394log(tD) = 0.063 for l0 = 900 bp. For the data shown in tD) = 0.1 for l0 = 100 bp, and \u0394log(tD) = 0.03 for l0 = 900 bp. The inability to easily and quickly discriminate the 900 bp DNA from the 1000 bp DNA demonstrates the practical limits set on Mode I sample identification according to the size of the insertion or deletion that must be detected.We first experimentally illustrate the practical length resolution of the nanopore platform for identifying sample length and composition. We analyzed samples containing mixtures of DNA fragments composed of one or two well-defined lengths. The resulting event diagrams create unique fingerprints that can be used to distinguish different lengths of DNA (Mode I) or whether or not a fragment of DNA has been cut (Mode II). Fig C in . CompariFig D in . Howeveriate l0  from l01000 bp, , since t1000 bp, . ReturnitD or IB distribution . To quantitatively identify unknown samples, which must fall into one of two (or a few) known possible genetic variation classifications at each relevant locus, we have developed a classification framework for nanopore sensing based upon Bayesian classification [We seek to develop a statistically robust and data processing procedure for nanopore-based discrimination among DNA fragment lengths. We note that in diagnostic tests, biochemical assays are ideally designed to classify samples among just a few known possible cases, rather than to produce a continuously-valued scalar associated with a physical quantity of the system  to the event diagram to describe each distribution as a sum of one or more two-dimensional covariant Gaussian probability density functions (p(\u03b8model|Z) is the GMM for case Z for the model data set \u03b8model (here: variables IB and tD) summed over all k components, wk is the relative weighting of each component such that \u03bck is the mean of each component, and \u03a3k is the covariance matrix of each component. We then calculate the posterior likelihood, p(Zi|\u03b8), that an unknown sample translocation data set, \u03b8, belongs to the a priori distribution Zi according to Bayes\u2019 rule (The event diagrams shown in unctions .p(\u03b8modees\u2019 rule :p(Zi|\u03b8)Zi yields the higher posterior likelihood. The posterior likelihood also represents the expected accuracy of the resulting decision. Here, the prior probabilities are taken to be equal, P(Zi) = 0.5. In a clinical application, the prior probabilities may be adjusted to account for the known incidence of each possible case in the clinical population under consideration, and additional weighting factors may be included to bias the classifier and thereby avoid costly diagnostic errors.The \u201cunknown\u201d sample is then assigned to whichever N. p(A|\u0398) or p(B|\u0398) for correctly assigning an unknown sample to either case A  or case B  for N translocations of the unknown sample. Each light gray curve represents a single simulation run of the posterior probabilities calculated after N translocations of the unknown sample, using a randomly selected training set of 1500 translocations for the GMM fit, and a randomly selected and ordered test data set from the remaining translocations . The red and blue curves represent the average posterior probabilities for correctly identifying an A sample and a B sample, respectively, averaged over all 1000 simulation runs. These clearly highlight a general trend emerging from our data: a rapid increase in the probability for N of just a few tens of events from the unknown test sample, followed by a slow increase for larger values of N.The confidence with which an unknown sample may be classified increases with the number of translocations collected, M of translocations used for fitting the GMM. A and B for a range of model data set sizes, M. It is clear that larger training sets are necessary to achieve high levels of confidence; in this case, if fewer than M = 700 samples make up the training sets, the confidence level of correctly identifying the cut fragments does not rise above 95%, even for relatively large N = 500 translocations. However, the number of points M required in the model set varies widely depending upon the similarity of the distributions to be discriminated: for example, for the distributions shown in M = 100 data points are necessary to exceed 95% confidence with only N = 50 test points  less than a minute would be sufficient to obtain a confident result. Additionally, the curves shown in A (1000 bp) rather than B (900+100 bp).The rate of convergence and maximum confidence of the posterior probability curves shown in Fig G in . Insets S. aureus, in which a SNV confers antibiotic resistance via the parC gene, and M. tuberculosis, in which a SNV affects virulence via the mazG gene. The emergence of methicillin-resistant S. aureus (MRSA) strains in recent years has increased the incidence of severe nosocomial and community-acquired infections. The wide variation in antibiotic resistance across this set of pathogens necessitates either immediate treatment with broad-spectrum antibiotics that can perpetuate the evolution of resistant strains, or accurate but expensive and time-consuming pathogen characterization prior to treatment, or (often) both. MRSA exhibits a relatively high degree of genetic variation due in large part to frequent horizontal gene transfer [M. tuberculosis, the bacterium responsible for tuberculosis (TB), exhibits relatively little genetic diversity among its various strains [We demonstrate here the highest sensitivity of our proposed analysis by discrimination of critical single nucleotide variations in actual pathogen genomes, selected at sites that are known to alter pathogenicity. We selected two model pathogens with very different characteristic phenotypic effects caused by a lone SNV: transfer . Antibiotransfer . MRSA altransfer . In cont strains  but has S. aureus, we selected two closely related isolates of the well-characterized USA300 strain, USA300-FPR3757 and USA300-HOU-MR. Both of these isolates are methicillin-resistant, but otherwise possess very different spectra of antibiotic resistance [M. tuberculosis, we selected closely related virulent (H37Rv) and avirulent (H37Ra) strains, which differ only in several key SNVs and indels [M. tuberculosis, and H37Ra is even used to boost immunogenicity during TB immunization.For each of these two prototypical pathogens we have selected two well-studied and closely related strains to illustrate the sensitivity of our technique by sensing single critical SNVs that differentiate the pathogens. For methicillin-resistant sistance , 23 due d indels . This pamazG gene in M. tuberculosis encodes an NTP pyrophosphohydrolase, which promotes cell viability under oxidative stress [mazG at location 239 bp in H37Ra is believed to be critical to the original emergence of this less virulent strain  by conferring a competitive advantage during aging-mediated cell lysis [mazG gene and its subsequent digestion by running native PAGE gels, as shown in M, these M. tuberculosis strains can be distinguished with >99.5% confidence in N = 50 translocations or fewer, using a model data set size of only ~100 points  and amplified a subregion that includes a critical SNV site. While both strains are methicillin resistant, the FPR3757 shows a much larger spectrum of antibiotic resistance [For the MRSA strains, we chose the sistance . SpecifiA critical aspect of the approach outlined here is careful sample design to allow efficient sample identification at each site under consideration. Both Mode I and Mode II sensing depend upon differences in translocation time and blockage level caused by differences in sample length. In addition to PCR design of sample length, these variables are controlled by a multitude of factors, including pore geometry, material, and functionalization, electrical bias, and buffer conditions. Mode II sensing is additionally dependent upon the location of the restriction site within a fragment; asymmetric sites may produce a more obvious change in translocation dwell time or in blockage level, but very short fragments may produce dwell times below the sensing resolution of the nanopore, leading to \u201cmissed\u201d events. These factors can be balanced for any given nanopore system to enable both Mode I and Mode II sensing and minimize the possibility for missed events. Note that for Mode I type sensing, the restriction digest is eliminated, conserving both time and resources. However, Mode II type sensing is more suitable to discriminate critical single nucleotide variations, which alter pathogenicity, and also proves the high sensitivity of our method.Solid-state nanopore based biosensing is a rapidly growing field due to its practical and conceptual simplicity, portability and versatility. To date, few reports have demonstrated the utility of the method towards clinical diagnostic applications. Yet as we have shown here, nanopores are well-suited to make statistically robust diagnostic classifications among different DNA lengths with real single-molecule data, even in cases where the distributions significantly overlap. Utilizing a Bayesian statistical model, we have demonstrated that nanopore sensing can be used to discriminate among pathogens based on well-known genomic variations. Both large indels (Mode I) or short indels and single nucleotide variations (Mode II) can be targeted using proper sequence-specific digestion with off-the-shelf restriction enzymes. Furthermore, the Bayesian classifiers indicate the statistical confidence of each classification as a function of the number of nanopore events obtained in each measurement. Even at this preliminary stage of development we find that only a few tens of events (obtained in just a few minutes using a single pore) are sufficient to produce a statistically reliable result with well-defined and small error margins.Our method is general and can be adapted to address many different \u201cmultiple-choice\u201d clinical questions using a nanopore biosensor or other single molecule approaches. Future extensions of this work may seek to design and implement large panels of critical sites that represent the minimum sets necessary to characterize genomic variation for various applications in healthcare and research, and to develop additional sensing modalities. Although the primary design challenge currently remains linked to the location and availability of restriction digestion sites, we expect that the ongoing development of designer restriction enzymes, for example systems based on modular zinc fingers , TALENs The nanopore fingerprinting approach presented here addresses clear needs in clinical molecular diagnostics for a rapid and simple sensor that can identify a wide range of genomic variation in pathogens to inform treatment options. We have shown here discrimination of both large and small scale genomic variations between pathogen strains, down to single SNVs. The large, flexible sample design space for lengths, cut sites, and enzyme selection at each critical locus ensures that the technique is highly customizable for different genomic variation panels that could profile pathogenicity, antibiotic resistance, or even sequence type. The inherent scalability, minimal sample requirements, speed, and simple readout of the nanopore platform would all facilitate on-site and perhaps even automated use: As successive events are recorded, an increasingly clear fingerprint of translocation times and blockage levels will permit online software to \u201ccall\u201d the sample as soon as enough events have been accumulated. Our technique is highly portable and customizable, and the binary data would be readily transferrable among different labs.2 (500 nm) and low-stress amorphous silicon nitride . The SiNx was locally thinned to <10 nm (1.5\u20132 \u03bcm circular wells) using a controlled RIE etch. Freestanding membranes of SiNx (60x60 \u03bcm) were created by through-etching the wafer with KOH, with the locally etched wells aligned to the etched freestanding SiNx membranes. Nanopores were fabricated in the thinned SiNx regions using a high resolution TEM (Jeol 2010F), as previously reported [Nanopore chips were fabricated on a 4\u201d silicon wafer coated with SiOreported . Pore foM. tuberculosis strains H37Ra (ATCC\u00ae25177) and H37Rv (ATCC\u00ae25618) were freshly obtained from American Type Culture Collection (ATCC). In order to obtain the mazG gene from both M. tuberculosis strains, we designed primers for PCR amplification  using New England Biolabs Phusion polymerase. The same protocol was used for both strains using the same primers. We chose a restriction enzyme specific to the single nucleotide variation: for the mazG gene in H37Rv, NaeI (New England Biolabs) will cut the amplicon into two pieces of 321 and 621 bp, whereas the mazG gene from H37Ra will not be digested with this enzyme (942 bp). Two different strains of methicillin-resistant S. aureus  were used in order to distinguish a single nucleotide variation in the parC gene. The amplicon for the MRSA parC gene was digested with the restriction enzyme BseRI (New England Biolabs), which cut the HOU-MR fragment into two pieces (245 bp and 640 bp), while the FPR3757 fragment remained full-length (885 bp). PCR for the MRSA strains was performed under similar conditions as for the M. tuberculosis gene amplification. Sequences and further details for these specific genes and corresponding PCR primers are provided in 100 bp, 900 bp and 1 kbp, dsDNA was purchased from Fisher Scientific (NoLimits\u2122 DNA) and used without further purification. Electrical NP measurements were performed in a dark, double-insulated Faraday cage using our custom cell (described elsewhere) . The iondataA and dataB to represent a model set  a test set . 2-D Gaussian mixture models (Afit and Bfit) with either one (uncut fragment) or two components (cut fragments) were fit to each model set by expectation maximization, yielding fit parameters \u03bc (component means), \u03a3 (component covariance matrices), and w (mixture weights for each component). Posterior probabilities p(Afit|\u0398A) (correct ID of \u0398A as type A data), p(Bfit|\u0398B) (correct ID of \u0398B as type B data), p(Bfit|\u0398A) (incorrect ID of \u0398A as type B data), and p(Afit|\u0398B) . Reported posterior probabilities are the average of many  iterations of this method. For each iteration, randomly selected disjoint subsets were selected from the original data sets S1 FileM. tuberculosis and methicillin-resistant S. aureus SNV detection: Additional data and numerical simulations . Fig A in S1 File. PCR and Restriction Digest Products. Native PAGE showing successful PCR amplification of the mazG gene (M. tuberculosis) and parC gene (S. aureus). Digestion reactions yield either cut or uncut amplicons depending upon the parent strain. Lane 1: loading dye. Lane 2: 100 bp NEB ladder. Lane 3: mazG gene amplified from H37Ra (942 bp). Lane 4: mazG gene amplified from H37Rv (942 bp). Lane 5: mazG from H37Ra after digestion with NaeI enzyme, not cut. Lane 6: mazG from H37Rv after digestion with NaeI, cut into two fragments of 321 and 621 bp. Lane 7: parC gene fragment amplified from HOU-MR strain (885 bp). Lane 8: parC gene fragment amplified from FPR3757 strain (885 bp). Lane 9: parC from HOU-MR after digestion reaction with BseRI, cut into two fragments of 245 and 640 bp. Lane 10: parC from FPR3757 after digestion reaction with BseRI, not cut. Digestion reactions were performed at 37\u00b0C for 1hr in NEB Cutsmart buffer. 10 units of enzyme were used for each digestion reaction. Fig B in S1 File. Gaussian Mixture Model Fits for DNA Translocation. Gaussian mixture model fits to translocations of single-length DNA samples through a 4.8 nm diameter nanopore . (a) 100 bp NoLimits DNA. (b) 200 bp NoLimits DNA. (c) 900 bp NoLimits DNA. (d) 1000 bp NoLimits DNA. Raw tD and \u0394I data are shown in Fig C in S1 File. Bayesian Posterior Estimates for Nanopore Sample Identification. Bayesian posterior estimates p(100bp|\u0398) and p(1000bp|\u0398) for test data sets of N points given a model based on M points. Data is bootstrapped from translocations of (a) 100 bp NoLimits DNA and (b) 1000 bp NoLimits DNA  and p(200bp|\u0398) for test data sets of N points given a model based on M points. Data is bootstrapped from translocations of (a) 100 bp NoLimits DNA and (b) 200 bp NoLimits DNA  and p(1000bp|\u0398) for test data sets of N points given a model based on M points. Data is bootstrapped from translocations of (a) 900 bp NoLimits DNA and (b) 1000 bp NoLimits DNA  1000 bp at 1 nM. (b) 1:1 ratio of 800 bp + 200 bp, total concentration 2 nM. (c) Gaussian mixture model fit, 1000 bp. (d) Gaussian mixture model fit, 800 bp + 200 bp. (e) Bayesian posterior estimate p(1000bp|\u0398) for test data sets of N points given a model based on M points. (f) Bayesian posterior estimate p(800+200bp|\u0398) for test data sets of N points given a model based on M points. Translocations for all samples were collected in a single nanopore  with a +300 mV bias relative to trans (open pore current: 13 nA). To facilitate visualization of population density, a random white noise offset below the acquisition rate of this data  has been added to each tD in panels (a) and (b). Numerical simulations for panels (e) and (f) were bootstrapped from the data in panels (a) and (b), respectively. Each point represents the average of 1000 simulated posterior estimates, each of which uses randomly selected (disjoint) model set M and test set N. M. tuberculosis H37Ra vs. H37Rv mazG Samples.Fig G in S1 File. Identification of  Bayesian posterior estimates p(H37Ra|\u0398) and p(H37Rv|\u0398) for test data sets of N points given a model based on M points. Data is bootstrapped from translocations of (a) Tuberculosis H37Ra and (b) H37Rv mazG restriction digested fragments as described in S1 File Sections 1 and 2. Each point represents the average of 1000 simulated posterior estimates, each of which uses randomly selected (disjoint) model set M and test set N. S. aureus FPR3757 vs. HOU-MR parC Samples.Fig H in S1 File. Identification of  Bayesian posterior estimates p(FPR3757|\u0398) and p(HOU-MR|\u0398) for test data sets of N points given a model based on M points. Data is bootstrapped from translocations of (a) MRSA FPR3757 and (b) HOU-MR parC restriction digested fragments as described in S1 File Sections 1 and 2. Each point represents the average of 1000 simulated posterior estimates, each of which uses randomly selected (disjoint) model set M and test set N.Gene and primer sequences for tuberculosis and MRSA strains. Additional details for PCR and restriction digest . Bayesian classification for Mode I: Additional data and numerical simulations . Bayesian classification for Mode II: Additional data and numerical simulations . (PDF)Click here for additional data file."}
+{"text": "Phenobarbitone is the most common first-line anti-seizure drug and is effective in approximately 50% of all neonatal seizures.To describe the response of electrographic seizures to the administration of intravenous phenobarbitone in neonates using seizure burden analysis techniques.-1) was compared to seizure burden in periods of increasing duration after each phenobarbitone dose had been administered . Differences were analysed using linear mixed models and summarized as means and 95% CI.Multi-channel conventional EEG, reviewed by experts, was used to determine the electrographic seizure burden in hourly epochs. The maximum seizure burden evaluated 1 h before each phenobarbitone dose (T+1) . The percentage reduction was 74% (IQR: 36-100). This reduction was temporary and not significant within 4 h of administrating phenobarbitone. Subgroup analysis showed that only phenobarbitone doses at 20 mg/kg resulted in a significant reduction in the maximum seizure burden from T-1 to T+1 (p = 0.002).Nineteen neonates had electrographic seizures and met the inclusion criteria for the study. Thirty-one doses were studied. The maximum seizure burden was significantly reduced 1 h after the administration of phenobarbitone (TPhenobarbitone significantly reduced seizures within 1 h of administration as assessed with continuous multi-channel EEG monitoring in neonates. The reduction was not permanent and seizures were likely to return within 4 h of treatment. Seizures are harmful to the developing neonatal brain , are a nThe development of ASDs for neonates remains a challenging area due to developmental differences between the neonatal and adult brain such as higher concentrations of intracellular chloride and a lower expression of \u03b3-aminobutyric acid (GABA) receptors in the developing neonatal brain . EvidencAs part of an ongoing study of neonatal seizures, neonates were enrolled from the neonatal intensive care units in Cork University Maternity Hospital (Ireland) and University College London Hospital (United Kingdom) from January 2009 to October 2011. Neonates \u226537 weeks\u2019 gestation were enrolled for EEG monitoring if there was any evidence of encephalopathy or seizures within 72 h of age. Neonates who had at least one ASD dose administered during electrographic seizures were included in the study. Neonates were excluded if all their phenobarbitone doses were administered without electrographic evidence of seizures. Institutional review board approval was obtained from the Clinical Research Ethics Committees of the Cork Teaching Hospitals (Ireland) and the National Health Service in the United Kingdom via the Integrated Research Application Service. Written, informed consent was obtained from at least one parent of each neonate who participated in this study.All clinical seizures and EEG seizures recognized by the clinical team were treated. The standardized protocol for ASD usage was similar in both hospitals. At the discretion of the attending neonatologist, a phenobarbitone loading dose of 10 or 20 mg/kg was administered intravenously on seizure recognition. Subsequent phenobarbitone doses up to an accumulated dosage of 40 mg/kg or second- (intravenous phenytoin or midazolam), third- and fourth-line ASDs were administered if clinical and/or electrographic seizures recurred. The time, dose and accumulated dosage of phenobarbitone were recorded. We assumed a zero clearance rate when calculating the accumulated dosage as the majority of doses were given well within the half-life of phenobarbitone ,11,12. A-1) was compared to a time period of 1 h in duration beginning immediately after cessation of the phenobarbitone infusion which was completed in 30 min. The MSB in time periods of increasing duration was also used to assess the duration of the effect of phenobarbitone administration. Time periods were increased from 1 h (T+1), in hourly increments  until the last electrographically recorded seizure (T+LR). For example, T-1 is the time period from 1 h before the start of phenobarbitone infusion until the start of phenobarbitone infusion, and T+3 is the time period from after the cessation of phenobarbitone infusion until 3 h after the cessation of phenobarbitone infusion  in each 1-hour period of the EEG recording based on the electrographic seizure annotations. This was defined as the accumulated seizure duration within a 1-hour window, shifted across the EEG monitoring period with a 1-min interval . Linear mixed models with a neonate-level random effect were used to account for possible correlations among observations from the same neonate (more than one phenobarbitone dose per neonate). For comparisons between groups, group was included as a fixed effect in the linear mixed model. Results based on linear mixed models were presented as means (95% CI). The comparison between MSB before and after 1 h (T-1 vs. T+1) was also performed in subgroups defined by dosage and accumulated dosage. We denote the number of neonates as nn and the number of doses as nd. All statistical analyses were performed in SAS 9.3 , and p < 0.05 was considered as statistically significant.Continuous variables were described using medians [interquartile ranges (IQR)] and categorical variables using frequencies. Differences between MSB before and after phenobarbitone administration were calculated for each period . Therefore, the effectiveness of phenobarbitone was measured in the remaining 19 neonates; table During the study period, of the 35 neonates with electrographic seizures identified, 16 did not meet the inclusion criteria for ASD analysis . The median (IQR) time between seizure onset and phenobarbitone administration was 3.3 h . A significant MSB reduction was seen in the hour immediately after phenobarbitone administration . In fact, the MSB was not significantly reduced after phenobarbitone by T+4 (table +1)  compared to doses which did not completely reduce the MSB . Ten of the 13 doses which resulted in complete seizure reduction in T+1 were a first dose  vs. 10 mg/kg (nd = 11): \u221218.6 min/h  vs. \u22124.4 min/h ; p = 0.002]. In fact, 20 of 20 (100%) doses of phenobarbitone at 20 mg/kg resulted in a reduction in MSB during T+1, and in 13 of 20 (65%) doses the MSB was zero during T+1. This result is reflected when observing the effect of accumulated dosage, as the MSB reduction in T+1 was significantly higher for accumulated doses of 20 mg/kg  and 40 mg/kg  compared to an accumulated dose of 30 mg/kg [mean difference (95% CI): \u22121.0 min/h  (nd = 6); p = 0.001 and 0.017, respectively (fig. The MSB reduction was greater when 20 mg/kg was administered compared to a dose of 10 mg/kg [mean difference (95% CI) of 20 mg/kg . Additional details on these ASDs can be found in the supplementary material at www.karger.com/doi/10.1159/000443782.A total of 13 doses of additional ASDs were given to neonates who received phenobarbitone as a first-line ASD . Only 1 dose of phenobarbitone at 20 mg/kg was administered after the administration of second-line ASD (<1 h after phenytoin). No second- or third-line ASDs were given in TWe have shown that a loading dose of phenobarbitone results in an immediate but temporary reduction in electrographic seizure burden in most term neonates; seizures returned to pre-treatment levels within 4 h of administration. We have also shown that 20 mg/kg doses were more effective than 10 mg/kg, and that phenobarbitone was more likely to abolish seizures in the short term if given before a large accumulation of seizures was apparent.A receptors [+1 in 65% of cases when the dose is 20 mg/kg. Conflicting results between animal and clinical studies must be resolved in order to develop improved treatment strategies for neonatal seizures.Phenobarbitone, a barbiturate, primarily has an inhibitory effect in the adult brain by prolonging the action of GABA, acting mainly on the GABAeceptors . The pureceptors ,17. Thiseceptors . This sueceptors  and pheneceptors ,4. We haA receptor subunit, activation of a non-functional \u2018spare\u2019 GABAA receptor, uncoupling of receptors and post-translational modification of GABAA receptors have been hypothesized as possible mechanisms for this pharmacoresistance [We have shown that the effectiveness of phenobarbitone is temporary and the reduction in seizure burden is limited beyond 4 h of administration. This is conspicuously shorter than the pharmacokinetic half-life of phenobarbitone (range: 45-500 h  and is vsistance . We havesistance  hypothesSeizures are intermittent and highly variable, and show a natural tendency to decay after a long period of time . Up to 8In maternity units like ours, which monitor neonates with seizures intensively, we still found suboptimal treatment of neonatal seizures. If electrographic seizures emerged during out-of-hours periods, there were no alarm systems to alert clinical teams to ongoing electrographic seizures , hence tPhenobarbitone immediately reduced the accumulation of seizures in a cohort of neonates with mixed aetiology. The effect was temporary and the reduction in seizure burden was not significant within 4 h of treatment. Doses of phenobarbitone at 20 mg/kg, rather than 10 mg/kg, were significantly more effective in reducing seizure burden. Phenobarbitone was also more effective if administered when the seizure burden was relatively low.None of the authors has any conflict of interest to disclose."}
+{"text": "TB) phase and heliconical tilted smectic C (SmCTB) phase are formed. The formation of a variety of helical structures is accompanied by a gradual freezing of molecular rotation. In the lowest temperature smectic phase, HexI, the twist is expressed through the formation of hierarchical structure: nanoscale helices and mesoscopic helical filaments. The short-pitch helical structure in the smectic phases is confirmed by resonant X-ray measurements.Chiral symmetry breaking in soft matter is a hot topic of current research. Recently, such a phenomenon was found in\u00a0a fluidic phase showing orientational order of molecules\u2014the\u00a0nematic phase; although built of achiral molecules, the phase can exhibit structural chirality\u2014average molecular direction follows a short-pitch helix. Here, we report a series of achiral asymmetric dimers with an odd number of atoms in the spacer, which form twisted structures in nematic as well as in lamellar phases. The tight pitch heliconical nematic (N Systems that form chiral structures from achiral molecules are not common. Here, the authors synthesise a compound consisting of asymmetric and achiral bent-shaped mesogens, which exhibit a variety of liquid crystal phases including one in which chiral structures form from achiral constituent molecules. Although it was to transpire that the new phase (later designated B4) reported in this work and described as a low dimensional LC system, was in fact a 3D crystal. However, the intense research stimulated by the Tokyo group\u2019s finding ultimately resulted in the discovery of the twist-bend heliconical nematic (NTB) phase3, some 35 years after its prediction by Meyer4. In the intervening period, Dozov independently predicted the existence of the helical nematic and smectic phases using symmetry arguments5. At the root of Dozov\u2019s prediction was the assumption that bent molecules have a natural tendency to pack into bent structures, however as uniform bend is not permitted in nature it must be accompanied by other local deformations, namely either splay or twist of the average molecular axis direction (director). The splay-bend nematic phase is achiral, by contrast, in the NTB phase the director forms a conical left- or right-handed helix. The NTB phase provided the first example of spontaneous chiral symmetry breaking in a fluid with no spatial ordering8. The helix in the NTB phase is extremely short, typically ~ 10\u2009nm (3\u20134 molecular distances)10. For the overwhelming majority of twist-bend nematogens, the NTB phase is preceded by a conventional nematic (N) phase with uniform director structure, for which the strongly bent molecules give rise to small values of the bend elastic constant8. In fact, there are just three examples of direct NTB\u2014isotropic phase transitions13. On cooling, the vast majority of NTB phases either crystallise or vitrify, and only rarely is a NTB-smectic phase transition observed, specifically either a smectic A phase typical for rod-like molecules , or a broken layer modulated B1 type phase16, typical for bent molecules have been observed. We have yet to establish and understand how the bent molecules will self-assemble into smectic phases if the bend elastic constant becomes anomalously low. Here we show that as in the nematic phase, such achiral bent molecules also spontaneously form short-pitch length helical structures in smectic phases.The spontaneous formation of chiral structures from effectively achiral molecules is at the heart of intense worldwide research, as the breaking of mirror symmetry is a fundamental issue in chemistry, physics and biology and plays a central role in the origin of biological homochirality. Twisted structures made from chiral building blocks are relatively common in nature, the archetypal examples being DNA or proteins that can exist in helical forms not only in solids but also in the liquid state, whereas examples of achiral molecules that assemble into helical aggregates are much less common, and until recently such helical aggregates were observed only for 3D ordered crystals. In the area of liquid crystals (LC), interest in chiral phases consisting of achiral building blocks began with the report by Sekine et al.17 and in an electrically tunable laser18.The simplicity of the twist-bend nematic phase has significant application potential, and a conventional nematic phase having a small bend elastic constant may itself be utilised in new technological applications such as the electrically controlled selective reflection of lightTB phase, can be realised using odd-membered mesogenic dimers. Such dimers are built of two rigid mesogenic moieties linked by a flexible spacer, normally an alkyl chain19. The liquid crystalline behaviour of dimers is strongly dependent on the length and parity of the spacer and this is most commonly attributed to how the average shape of the molecule is controlled by conformations of the spacer. Thus, if we consider the spacer to exist in an all-trans conformation then for an even-membered spacer the long axes of the mesogenic groups are essentially parallel and the molecule is linear, whereas for an odd-membered spacer they are inclined at some angle with respect to each other giving a bent molecular shape.The molecular curvature, that is essential for the formation of the NHere, we report the characterisation of a homologous\u00a0series of dimeric mesogenic molecules, which owing to the\u00a0odd-membered linkage adopt bent geometry. For the short homologues the phase transition between the uniform and heliconical nematic phases was found, whereas for longer homologues, a complex sequence of smectic phases was observed in which the tilted phases show a helical precession of the molecules around the cone axis, similar to chiral smectic phases. The lowest temperature smectic phase was found to exhibit structural chirality at different length-scales, i.e., layer chirality, nanoscale helices and mesoscopic helical filaments.n represents the number of carbon atoms in the terminal alkyl chain) is shown in Fig.\u00a0The molecular structure of the 4-[{[4-({6-[4-(4-cyanophenyl)phenyl]hexyl}oxy)phenyl] methylidene}amino]phenyl-4-alkoxy-benzoates . For longer homologues, the NTB phase is extinguished, and instead, up to four lamellar phases were found below the conventional nematic phase. The layer spacing in the smectic phases corresponds approximately to the full molecular length. The lowest temperature lamellar phase is the same for both, short and long, homologues, its X-ray pattern in the high-angle range shows a narrowed, split signal.Homologues with short terminal chains, 20. The characteristic XRD pattern obtained for an aligned sample, in which one of the high-angle signals is seen in an equatorial position with respect to the low-angle signals , XRD patterns with a single, broad high-angle signal were detected, indicating liquid-like positional ordering of the molecules within the smectic layers , in which molecular rotation around the long axis is to some degree frozen. The homeotropic optical texture of the tilted smectic C phase excludes its simple (uniplanar) synclinic or anticlinic lamellar structure, as both SmCs and SmCa phases are biaxial and form schlieren textures. The optical uniaxiality of the\u00a0tilted phase suggests an averaging of molecular orientations owing to the formation of a short helix\u2014we term the phase the\u00a0 twist-bend SmCTB  for CB6OIBeO7 saturates at ~\u200910 degree, and the same value of the tilt angle has been deduced from changes of the smectic layer spacing  at the carbon absorption K-edge, a technique used previously to probe the helical structure of the N0\u2009\u00c5 Fig.\u00a0. The pit4 phase, although it should be noted that filaments of B4 phase have an internal crystalline structure26. The origin of chiral optical effects in the HexI phase is not clear; for tilted smectics consisting of chiral molecules, it is known that optical activity becomes negligible if the pitch is much shorter than the wavelength of light. Therefore, optical activity and CD in the HexI phase should be attributed, as with the B4 phase, to the internal layer structure  induced by the strongly non-homogenous electron distribution across the layer and the tilted arrangement of the strongly biaxial molecules27. Lack of CD and optical activity in the SmCTB phase indicates that essentially free molecular rotation around the molecular long axis occurs, and therefore the biaxality is too weak to cause detectable \u2018layer chirality\u2019.The texture of the HexI phase in cells with homeotropic anchoring is very weakly birefringent Fig.\u00a0, and a sTB materials, was found for the studied compounds. Instead, only a weak change in the birefringence and a bi-polar response under high electric fields were detected, except for the HexI phase, which was not sensitive to electric field.No clear optical flexoelectric switching, reported previously for other NTB phases was found, whereas for longer homologues, as the tendency for the alkyl chains and aromatic moieties to nanoscale separate increases, a complex sequence of smectic phases was observed in which the tilted phases show a helical precession of the molecules around the cone axis, similar to chiral smectic phases. The heliconical smectic phase is preceded by the non-tilted SmA and SmAb phases. Apparently, on lowering the temperature rotation around the long molecular axis is gradually frozen and as a result the transition from the SmA to SmAb phase is observed. The gradual freezing of rotation is accompanied by a more pronounced layer spacing increase, owing to lower interdigitation of molecules between neighbouring layers and a stretching of the terminal chains. The lack of a polar response under an electric field shows that in the SmAb phase the dipole moments of the molecules are locally compensated. The X-ray studies revealed that upon lowering temperature, for intermediate length homologues the SmAb phase undergoes a transition (weakly first order or continuous) to a tilted phase, which surprisingly is optically uniaxial. This optical uniaxiality is inconsistent with a simple synclinic or anticlinic SmC phase structure, and indicates that averaging of molecular orientation must take place through the formation of a helioconical structure. The resonant X-ray scattering experiment indeed proves the existence of the superstructure, with a pitch corresponding to 3\u20134 smectic layers. This is the first report showing unambiguously that a short-pitch helical structure may be formed spontaneously in smectic phases consisting of achiral molecules. Such short helices were found previously for smectic phases comprising chiral rod-like molecules (SmC\u03b1 phase) as a result of competing interactions between nearest and next nearest layers\u2014the frustration in systems, in which next nearest interactions favour antiparallel tilt, is relieved by the formation of a helix that can be as short as a few smectic layers29. However the molecules studied here are achiral dimers and the helix formation is presumably driven by steric interactions arising from the bent molecular shape, in similar fashion to the NTB phase. The reported bent dimers have an extremely strong tendency to form twisted structures, this is evidenced not only by the heliconical arrangement of the molecules in the NTB and SmCTB phases, but also is expressed through formation of mesoscopic helical filaments in the lowest temperature smectic phase, the HexI phase.Summarising, for the short homologues of the CB6OIBeOn series the phase transition between the uniform N and heliconical N\u22121.Calorimetric studies were performed using differential scanning calorimeter (DSC) TA Q200, samples of mass 1\u20133\u2009mg were sealed in aluminium pans and kept in nitrogen atmosphere during measurement, both heating and cooling scans were performed with rate 10\u2009K\u2009mindSmC\u2009=\u2009dSmA cos(\u03b8), with respect to the layer thickness value extrapolated from the data recorded in the orthogonal SmA phase to the temperature range of the tilted phase.Wide angle X-ray diffractograms were obtained with a Bruker D8 GADDS system . Samples were prepared as droplets on a heated surface. The temperature dependence of the layer thickness was determined from small-angle XRD experiments performed with a Bruker D8 Discover system  working in reflection mode, homeotropically aligned samples were prepared as thin films on a silicon wafer. The tilt angle in the tilted smectic phase was calculated from the decrease of the layer spacing, TB and HexI phases, respectively. The X-ray beam with a cross-section of 300\u2009\u00d7\u2009200\u2009\u03bcm was linearly polarised, with the polarisation direction that can be continuously changed from the horizontal to vertical. Samples with thickness lower than 1\u03bcm were placed between two 100-nm-thick Si3N4 slides. The scattering intensity was recorded using the Princeton PI-MTE CCD detector, cooled to \u221245\u2009\u00b0C, having a pixel size of 27\u2009\u03bcm, with an adjustable distance from the sample. The detector was translated off axis to enable a recording of the diffracted X-ray intensity. The adjustable position of the detector allowed us to cover a broad range of q vectors, corresponding to periodicities from ~ 5.0\u2013300\u2009nm.The resonant X-ray experiment was performed on the soft X-ray scattering beamline (11.0.1.2) at the Advanced Light Source of Lawrence Berkeley National Laboratory. The X-ray beam was tuned to the K-edge of carbon strongest absorption peak, ~\u2009283.6\u2009eV, however for the studied dimeric material a few more peaks with different energy were also detected. An energy scan was done at two temperatures: 392 and 360\u2009K in the SmC\u03bb\u2009=\u2009546\u2009nm). The average retardation was measured in 10\u2009\u00d7\u200910 micron region, but space modulation of retardation was also evaluated with submicron resolution. For birefringence measurements the three micron cells were used with planar alignment layer. As the system allows the measurement of retardation only up to 273\u2009nm, the absolute value of retardation was determined therefore by comparing the results obtained for the samples having different thicknesses, 1.8 and 5 micron. The conical tilt angle in the SmCTB phase was deduced from the decrease of the birefringence with respect to the values measured in the SmA phase, \u0394nSmCTB\u2009=\u2009\u0394nSmA(3cos2(\u03b8)\u22121)/223, the birefringence of the SmA phase was extrapolated to the lower temperature range, assuming a linear temperature dependence of \u0394n in the SmA phase, the increase of the birefringence in the SmA phase on lowering temperature is due to the growing positional and/or conformational ordering of the molecules.Optical studies were performed using the Zeiss Imager A2m polarising microscope equipped with Linkam heating stage. Samples were observed in glass cells with various thickness: 1.8\u201310 microns. The microscope setup was equipped with an Abrio system for precise birefringence measurements. The birefringence was calculated from optical retardation of green light (\u22121 was applied using. Light transmission was monitored with PIN-20 photodiode mounted in microscope.Electrooptic response of the phases was studied in glass cells (1.6\u20135.0\u2009\u03bcm thick) covered with transparent ITO electrodes, and a surfactant layer ensuring planar sample alignment. Electric field of low frequency (<30\u2009Hz) and magnitude up to 30\u2009V\u2009\u03bcmk\u2009=\u20090.4 Nm\u22121 were used, the resonant frequency was in the range of 70\u201380\u2009kHz, and the typical scan frequency was 1\u2009Hz.AFM images have been recorded using a Bruker Dimension Icon microscope, working in tapping mode at the liquid crystalline-air surface. Cantilevers with a low spring constant, The CD spectra were collected with a J-815 spectropolarimeter from Jasco, Japan, using quartz plates as substrates for the samples.The data that support the findings of this study are available from the corresponding author upon reasonable request.Peer Review FileSupplementary Information"}
+{"text": "The experimentally observed phase transition sequence nematic (N)\u2009N\u2009+\u20096\u2009two-dimensional hexagonal (2D-H) is in good agreement with the theoretical predictions. Further, the present study also brings to light the effect of chirality on the N\u2009+\u20096 phase. In the chiral N\u2009+\u20096 phase the bond-orientational order within each \u201cpolymer\u201d bundle is found to be twisted about an axis parallel to the average polymer direction. This structure is consistent with the theoretically envisaged Moir\u00e9 state, thereby providing the first experimental demonstration of the Moir\u00e9 structure. In addition to confirming the predictions of fundamental theories of two-dimensional melting, these results are relevant in a variety of situations in chemistry, physics and biology, where parallel packing of polymer-like objects are encountered.We report the discovery of a thermodynamically stable line hexatic (N\u2009+\u20096) phase in a three-dimensional (3D) system made up of self-assembled  The occurrence of the hexatic phase was first proposed in the context of melting of two-dimensional 2D) crystals, as an intermediate phase between the crystalline and liquid phases1D crystal14line-hexatic or N\u2009+\u20096 phase since nematic order coexists with BO in the plane normal to Unlike their 2D counterparts, three-dimensional (3D) crystals melt directly into the liquid phase via a first-order transition. The exception however being 3D crystals made up of oriented polymer-like constituents where the possible presence of an intermediate hexatic phase has been theoretically demonstratedAlthough the N\u2009+\u20096 phase has been theoretically predicted to occur as an intermediate phase between the N and the 2D-H phases, this is not the case in the two systems where this phase has been experimentally reported until now. In both these systems the N\u2009+\u20096 phase arises due to packing frustrations imposed by the shape of the individual constituents, and occurs close to their highest concentration compatible with 2D packing.Occurrence of the N\u2009+\u20096 phase in 3D systems was first experimentally reported in dense aqueous solutions of long DNA fragments7In the other instance, occurrence of the N\u2009+\u20096 phase was reported in dense suspensions of thin hard colloidal discs that stack into long columnsMoir\u00e9 phase, consisting of braided bundles of polymers, with BO in each bundle twisted about an axis parallel to the average polymer direction; in this phase the twist axis is parallel to Influence of molecular chirality on the structure of the N\u2009+\u20096 phase has also been theoretically investigated141531718thermodynamically stable line-hexatic phase formed by self-assembled polymer-like micelles made out of a mixture of an amphiphile, sodium dodecylsulphate (SDS), and organic salt, p-toluidine hydrochloride (PTHC) ; (b) chiral analogue of N\u2009+\u20096 phase; and (c) the observation of the theoretically predicted N\u2009In this article we report the discovery of (a) a perpendicular geometry, where the incident x-ray beam is normal to q\u22a5, indicating that the cylindrical micelles have long-range orientational order along z. The position of the peak gives the average lateral separation between the micelles, while its width is inversely proportional to the translational correlation length \u03be in the medium. The intensity profiles at different temperatures  patterns obtained using samples in the eratures , reveal tructure .schlieren texture in the POM micrograph which is characteristic of the N phase . Micrographs obtained at different temperatures are shown in  N phase . On heat N phase , no indiperature  can be aparallel geometry with the incident x-ray beam parallel to q\u22a5 plane. In contrast, long-range BO in the high temperature (50\u2009\u00b0C) 2D-H phase gives rise to six sharp spots in the q\u22a5 plane  phase develops only after about 10\u2009hours, presumably due to the very high viscosity of these samples. Two transitions, indicated by abrupt changes in the texture, are seen at around 33\u2009\u00b0C and 40\u2009\u00b0C on heating; which correspond closely to the boundaries of the region highlighted in \u03be varies strongly with temperature. These transitions are reversible on cooling. SAXS peak width of the chiral sample shows identical temperature dependence as that of the achiral sample  and the total surfactant weight fraction of 0.5 (SDS\u2009+\u2009PTHC)/(SDS\u2009+\u2009PTHC\u2009+\u2009water)\u2009=\u20090.5)30Sodium dodecylsulphate (SDS) (>99% purity) and p-toluidine hydrochloride (PTHC) (98% purity) were obtained from Sigma-Aldrich. Experiments were performed at a PTHC to SDS molar ratio of 0.1  provided with a heating or cooling stage (Mettler FP82HT) and a central processor (Mettler FP90). Appropriate amount of samples were taken between a glass microscope slide and a glass coverslip. They were then placed inside the temperature-controlled chamber (heating or cooling rate 1\u2009\u00b0C/min) and observed between crossed polarizers. The temperature of each sample was varied from 25 to 60\u2009\u00b0C.\u22121. Temperature stability was better than 0.1\u2009\u00b0C. Samples for SAXS studies were taken in sealed glass capillaries. All the samples were initially aligned in the nematic phase and the alignment was found to be retained on heating to the higher temperature phases. Orientation of the average micellar axis, i.e. the nematic director SAXS experiments were carried out using a Hecus SAXS-Eye system, in conjunction with either a CCD detector or a position sensitive detector. Details about the experimental set up and data acquisition have been described quite elaborately elsewhereHow to cite this article: Pal, A. et al. Observation of the Chiral and Achiral Hexatic Phases of Self-assembled Micellar polymers. Sci. Rep.6, 32313; doi: 10.1038/srep32313 (2016)."}
+{"text": "The material FDO11DFCB3 (compound 2 in this work) remains the only example of a liquid-crystalline material to exhibit a phase transition from the heliconical twist-bend phase into a lamellar smectic A mesophase, additionally this material exhibits a previously unidentified mesophase. We have prepared and characterised several homologues of this compound, with each material subjected to an in-depth analysis by optical microscopy, calorimetry and small angle X-ray scattering studies. Despite FDO11DFCB3 being similar in chemical structure to the novel materials presented herein its liquid-crystalline behaviour is rather different, indicating an unexpected sensitivity of the twist-bend phase to molecular structure. TB) in liquid-crystalline dimers21 and later bent-cores22 and oligomers26 has given impetus to the study of mesomorphic materials of unusual molecular architecture. Several aspects of the NTB phase have been reviewed recently29, and the consensus has been that the local structure of the phase is helical with a pitch length in the region of 10\u2009nm31. This outcome, however, has been disputed33, and alternate models proposed34. Furthermore it has been clearly demonstrated that the incidence of the twist-bend phase is not especially sensitive to chemical composition36, with the only prerequisite being that a material is sufficiently bent \u2013 with the degree of bend having some control over the (relative) thermal stability of the phase35. When the twist-bend phase is chiral other \u2018nematic-like\u2019 mesophases have been reported whose structure is as-yet-unknown37, whereas a nematic-to-nematic transition has also been recently reported for a polar rod-like compound38.The discovery of the twist-bend phase (N2 in this work) and this remains the only example of the phase sequence N-NTB-SmA that we are aware of, with the identifications of both the N, twist-bend and SmA phases confirmed by miscibility39. This phase sequence presents something of a unique testbed for theoretical treatments of the twist-bend phase; however compound 2 is far from ideal for study because the phases are both monotropic and exhibited only over a short temperature range. In a related study we demonstrated that the smectic A phase was suppressed when either mesogenic unit was replaced with a number of different groups .Previously we reported the synthesis and characterisation of FDO11DFCB3 -2\u2032,3-difluoro-4\u2032-hydroxybiphenyls (i-1 \u2013 i-4) were available in house, other chemical intermediates were obtained from commercial suppliers. The intermediate i-5 was prepared as described previously39. Williamson etherification of i-1 \u2013 i-4 with i-5 afforded compounds 1\u20134, as shown in Fig.\u00a040. Small angle X-ray diffraction was performed using a Bruker D8 Discover equipped with a temperature controlled, bored graphite rod furnace, custom built at the University of York. The radiation used was copper K\u03b1 (\u03bb\u2009=\u20090.154056\u2009nm) from a 1 \u03bcS microfocus source. Diffraction patterns were recorded on a 2048\u2009\u00d7\u20092048 pixel Bruker VANTEC 500 area detector set at a distance of 121 mm from the sample, allowing simultaneous collection of small-angle and wide-angle scattering data. Samples were filled into 0.9 mm OD glass capillary tubes and aligned with a pair of 1\u2009T magnets with the field perpendicular to the incident X-ray beam. Diffraction patterns were collected as a function of temperature (controlled to an accuracy of +/\u22120.1\u2009\u00b0C). Two dimensional diffraction patterns were radially averaged (0.05\u00b0 step size) to give one-dimensional profiles of scattered intensity as a function of two-theta, conversion into d-spacing (\u00c5) or scattering vector (Q) is then trivial.The intermediate 1\u20134 was studied by a combination of polarised optical microscopy (POM), differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS). Transition temperatures and associated enthalpies of transition were determined by DSC and are presented in Table\u00a0The liquid-crystalline behaviour of compounds 2 exhibits the phase sequence N-NTB-SmA with an additional and as yet unidentified smectic phase (SmX)39. If the length of the terminal alkyl chain of the 4\u2032--2,3-difluorobiphenyl mesogenic unit (CDFB) is shortened (1) or increased (3 and 4) then the NTB phase is not observed. All four materials exhibit N, SmA and unidentified SmX phases with compound 3 also exhibiting an additional as-yet unidentified SmY phase. The SmX-SmY transition is first order and occurs with a vanishingly small associated enthalpy, suggesting the two phases are closely related in structure.As shown previously, 1\u20134 was trivial based on their schlieren and focal-conic defect textures respectively, whereas the identification of the twist-bend phase for compound 2 was demonstrated previously. The SmX phase of 3 has a higher thermal stability than for 2 (or for that matter 1 or 4), with this material also exhibiting an additional unknown \u2018SmY\u2019 mesophase. The SmX phase was identified as a smectic C analogue based on the appearance of a schlieren texture in optically extinct regions of the SmA phase 36. Further cooling of the SmCA phase into the \u2018SmY\u2019 phase  or it is a new mesophase.The identification of the nematic and smectic A phases of ase Fig.\u00a0. As the ase Fig.\u00a0, the sch1\u20134 by small angle X-ray scattering. The aim of this study was to confirm (or refute) the assignment of the smectic mesophases exhibited by 1\u20134, and determine the subtype of the smectic A phase. Representative 2D SAXS patterns are given in Fig.\u00a0We now turn to the study of compounds 3 below the temperature at which the SmCA-\u2018SmY\u2019 transition occurs we did not observe any change in the 2D SAXS pattern  can be ruled out due to the lack of sharp scattering at wide angles, which would be indicative of the long in-plane correlation lengths present for such a phase. Instead the wide-angle scattering is diffuse and therefore indicative of a lack of in-plane ordering. However, this does not rule out the possibility of a SmI or SmF phase with long range bond-orientational order, which would show a similarly diffuse wide-angle scattering pattern. The vanishingly small enthalpy associated with the transition \u2013 coupled with the SAXS patterns \u2013 means that we feel the most likely explanation is that this is a transition between two subtypes of SmC phase, the higher temperature phase being SmCA. Whilst we are not able to definitively identify the lower temperature phase presently, transitions between different subtypes of the SmC phase (such as modulated smectic C phase (anti)) are known. More speculatively, this phase could be an example of a smectic twist-bend phase (SmTB?) although we concede that such a state of matter is hypothetical presently.Despite cooling compound ern Fig.\u00a0, the peasee Fig.\u00a0 both 2- i.e. the ratio between the d-spacing of the small angle scattering peak and the molecular length), allowing us to compare materials of differing length. Molecular lengths were calculated on isolated molecules in their all trans geometries at the B3LYP/6-31\u2009G(d) level of DFT . Clearly this neglects the flexibility that is inherent to a dimer with an undecamethylenedioxy spacer, however for the present use in the analysis of SAXS data it is sufficient and we will return to this point shortly.Rather than use the d-spacing of the small angle scattering peak we convert to a d/l ratio 41. However it would be surprising for a near 6\u2009\u00b0C increase in TNTB-N to be attributable to the rather weak aligning magnetic field used here (~1\u2009T). Secondly, as shown by the dashed line separating the nematic and twist-bend mesophases, there is a notable decrease in the d/l ratio across not only the entire NTB phase range but also in the nematic phase close to the transition. This reduction is a consequence of the molecules reorganising tilting away from the helical axis upon entering the heliconical twist-bend phase. Using the average d-spacing in the nematic phase range, we calculate that the conical angle reaches a maximum value of ~14\u00b0 immediately prior to the onset of the SmA phase. It appears that this tilt away from the director begins in the nematic phase, suggesting some pretransitional change maybe taking place close to the NTB-N transition. Following the NTB-SmA phase transition in compound 2, the diffuse small angle scattering peak is superseded by a sharp Bragg peak, and there is a significant increase in the d/l ratio, however the material crystallises prior to entering the SmC phase.A plot of the d/l ratio of 1, 3 and 4 are intercalated, with layer spacings of less than half a molecular length. The measured d/l ratios show a small dependence on the length of the terminal chain, with 4 having the largest layer spacing and 1 having the smallest. Only one major peak for layer spacing is observed in both smectic phases, and this strongly suggests there is no segregation of mesogenic units. The layer spacing in both the SmA and SmC phases is practically temperature independent (differing by only ~0.2\u2009\u00c5 over the entire temperature range of both phases in 3). SAXS data indicates that both smectic phases are intercalated monolayer phases, however in the case of the smectic C phase polarised optical microscopy indicates that the phase has anticlinic rather than synclinic layer organisation.Turning now to the other materials, both the smectic A and smectic C mesophases of 1\u20134 and are presented in Fig.\u00a01, 3 and 4 follow broadly the same trend over the entire temperature range, with the correlation length within the plane exhibiting only a marginal increase . The out-of-plane correlation length exhibits a continuous build up throughout the nematic phase range, but is effectively temperature independent in the smectic A and C mesophases. Compound 2 exhibits a twist-bend phase intermediate between the nematic and smectic A phases and consequently behavers somewhat differently to the other members of this homologous series. Throughout the nematic phase there is a steady build up in the correlation length parallel to the director, however upon entering the twist-bend nematic phase this decreases rapidly from ~150\u2009\u00c5 at the N-NTB phase transition to just ~70\u2009\u00c5 at the NTB-SmA phase transition. Upon entering the smectic A1 phase there is a marked increase in the out-of-plane correlation length . For all four compounds the in-plane correlation length remains small throughout the entire liquid-crystalline temperature range; this is clearly expected for the nematic and twist-bend phases, but in the smectic A and C phases this observation confirms the lack of positional organisation and refutes the possibility of higher ordered smectic phases being present.Determination of the full width half maximum of the major small- and wide- angle scattering peaks affords the correlation length parallel and perpendicular to the director. These were obtained for compounds et al.42 we obtained values of the orientational order parameter (S) for compounds 1, 3 and 4 as a function of reduced temperature across the whole phase range  upon entering the SmA phase, before decreasing marginally into the SmCA phase.Using the method of Davidson nge Fig.\u00a0. We coul1\u20134 exhibit smectic A phases given that they have a gross bent shape. As far as is known, all current examples of twist-bend to smectic phase transitions the lamellar phase is tilted36, and this observation is easily rationalised given the anticipated bent shape of these materials. Efforts to obtain single crystals of 1\u20134 suitable for structure determination by XRD were unsuccessful. We therefore opted to explore the conformational landscape of 1\u20134; replacement of the terminal chain with a methyl group reduces the complexity of the systems, however even using a simple trans/+ gauche/-gauche approximation the spacer alone has something like312 conformers. We therefore elected to study each individual torsional angle present in the spacer employing the AM1 semi empirical method using fully relaxed scans in 71\u2009\u00d7\u20095\u00b0 steps via the MODREDUNDANT keyword in Gaussian G09 e0140. From these the lowest energy conformation was identified, allowing us to plot \u0394E versus the resulting dihedral angle. For the \u2018first\u2019 and \u2018last\u2019 torsions  rather than to straighten the molecule out. For all torsions, with the exception of the \u2018first\u2019 and \u2018last\u2019 (i.e. adjacent to the mesogenic units) the all trans conformation was the energy minima, plots are given in the ESI to this article  which unfortunately precludes using selective 1D1H NOESY to obtain internuclear distances to support calculated geometriesAs noted in the introduction it is perhaps surprising that compounds see Fig.\u00a0 the tran Figures\u00a0 and SI203 \u2013 selected because of its relatively wide SmA phase range \u2013 doped with 0.5\u2009wt% hexadecyltrimethylammonium perchlorate was prepared and the sample filled into a glass cell with ITO electrodes . The behaviour in the nematic phase was unremarkable, with a Fr\u00e9edericksz transition observed at low voltage and frequency. At higher applied voltages and frequencies a rich array of electrohydrodynamic instabilities (Williams domains) were observed. Similar behaviour was observed previously for compound 2 and a detailed description of this is given previously39. Following cooling into the smectic A phase (~2\u2009\u00b0C below the N-SmA transition) we applied a voltage of 110\u2009V across the cell at low and high frequencies (1\u2009Hz and 1\u2009kHz respectively). For a typical low molecular weight SmA material (such as 8CB) a low frequency applied field would transform the active area of the cell into a scattering state, the subsequent application of a high frequency then returns this to a clear state46. In the case of compound 3 however, we did not observe any switching processes. It may be that as the smectic A phase (and indeed the SmC phases) are intercalated the layers are significantly harder to disrupt via application of external electric fields. This means that dimers and dimesogens that have intercalated mesophase structures are likely to be unsuitable for use in display devices utilising smectic phases.Given that the materials exhibit smectic A mesophases we performed preliminary studies into their potential use as hosts in smectic A based scattering devices. A mixture of compound 2 remains the only known example of a material exhibiting nematic, twist-bend and smectic A mesophases. In previous work we demonstrated that variations to the molecular structure of compound 2 typically retained the twist-bend phase but the smectic A phase was eliminated. In this present work we present several homologues of 2 in which the terminal chain length has been varied, and we find that for these materials the SmA phase is retained at the expense of the twist-bend phase. This has, however, allowed us to determine that the lower temperature \u2018X\u2019 phase of 2 (and its homologues) is an anticlinic C phase. Compound 3 also exhibits an additional smectic phase, whose structure is unknown, however evidence from POM, DSC and SAXS points to a SmC-type mesophase \u2013 this material therefore may exhibit an uncharted SmC \u2013 SmC transition.Compound 1\u20134 we observe that the behaviour of 1, 3 and 4 is largely the same; the smectic layer spacing indicates intercalated layer structure whereas the correlation length out-of-plane  shows a steady build up throughout the nematic phase before reaching values of ~300\u2009\u00c5 in the smectic A phase. Compound 2 displays somewhat different behaviour; the correlation length increases throughout the nematic phase range to a maximum of 150\u2009\u00c5 prior to the N-NTB transition, across the entire NTB phase range the out-of-plane correlation length decreases before reaching a value of ~300\u2009\u00c5 in the smectic A phase. Additionally the d-spacing value of the small angle scattering peak in the nematic phase decreases across the twist-bend phase range, and this is probably understood as a consequence of the molecules  tilting relative to the helical axis. For these compounds 1, 3 and 4 the high quality alignment obtained during SAXS study allowed us to obtain orientational order parameters; in the nematic phase the values are consistent with those reported previously for bent dimers47, whereas the values in the smectic A and C phases show a sharp increase at the phase transition. Finally, we note that intercalated smectic phases as found for compounds 1 to 4 (and related dimesogens) do not respond appropriately to applied electric fields, and therefore are of minimal use in display devices with the exception of use in formulating mixtures.In a detailed X-ray scattering study of compounds supplementary info"}
+{"text": "TB phases. This relationship demonstrates that molecular shape dictates the incidence of this fascinating phase of matter and leads us to speculate as to the existence of \u201ctwist\u2010bend nematic phases\u201d on length scales beyond those of the molecule.We prepared a significant number of unsymmetrical liquid\u2010crystalline dimers that exhibit the twist\u2010bend nematic phase; a state of matter that exhibits spontaneous breaking of mirror symmetry and, for some materials, a microsecond electrooptic response. A number of novel unsymmetrical bimesogens were synthesized and in comparing their thermal behaviour to previous literature examples, we have uncovered an unexpected relationship between the thermal stability of the nematic and N TB),TB phase, proposed independently by Meyer and Dosov,TB phase in bent\u2010core systems,The observation of a lower\u2010temperature mesophase lacking lamellar or positional molecular order ,TB phase, which we will now describe.It is well known that polar functional groups can, through dipole\u2010dipole or quadrupolar interactions, lead to antiparallel pairing of molecules; the extent to which this occurs is functional\u2010group dependent.1\u20136 has been reported previously.7\u201311 were synthesised, as shown in Scheme\u200512\u201316, which contain one polar and one apolar rigid unit, were prepared by the EDAC/DMAP\u2010mediated Steglich esterification between [4\u2010{9\u2010(4\u2010hydroxyphenyl)nonyl}phenyl 4\u2010cyanobenzoate] and a series of non\u2010polar benzoic and carboxylic acids, as shown in Scheme\u2005The synthesis and characterisation of compounds 1\u201311, which bear two polar \u201ccore\u201d units, were determined by a combination of polarised optical microscopy (POM) and differential scanning calorimetry (DSC). As shown in Table\u2005The transition temperatures of compounds 2, the NTB phases exhibited by 1\u201311 were monotropic, although the nematic phases of several materials  were enantiotropic. Compared to the symmetric parent material of the series, compound 1, the unsymmetrical materials all exhibit lower melting points, clearing points and nematic to NTB transition temperatures. Representative photomicrographs of the nematic and NTB mesophases of compounds 3, 7, 8 and 10 are given in Figure\u200510 is given in Figure\u2005TB\u2010N transition being weakly first order, as reported previously.With the exception of compound 1\u201311 was identified based on the characteristic textures  the associated enthalpies of both the N\u2010Iso and NTB\u2010N transition are highly reproducible, with only small deviations from the mean. Ultimately, and as reported previously,TB\u2010N transitions. In the case of compound 6, the associated enthalpies of both the N\u2010Iso and NTB\u2010N transitions were observed to decline with each successive heat/cool cycle, as shown in Figure\u2005With the exception of compound TB phase was correct, we studied compounds 1\u201311 by small angle X\u2010ray scattering (SAXS) and analysis of the conoscopic figures. As discussed by Zhu et\u2005al.,TB phase and the theoretically predicted splay\u2010bend nematic (NSB).TB phase in chiral dimers is optically positive, that is, the conical angle is less than the magic angle.10 and conoscopic figures are given for compound 7 in Figure\u20051\u201311 reveals that in all cases, the NTB phase is uniaxial; insertion of a \u03bb\u2010plate was used to demonstrate that the twist\u2010bend phase is optically positive in all cases and therefore, the conical angle is less than the magic angle.Q  was taken to exclude the possibility of the NTB phase being a splay\u2010bend modulated structure, as discussed by Zhu et\u2005al.To confirm whether the identification of the NTB\u2010N,12, 13 and 15 were undertaken primarily to see if further examples of the SmX\u2010NTB transition could be observed in unsymmetrical bimesogens with one polar and one apolar unit. By studying NTB to smectic phase transitions, it may be possible to gain significant insight into the twist\u2010bend nematic phase; however, we are aware of only four examples of such phase transitions.14, the incorporation of selenium will hopefully permit future study of the local structure of the NTB phase (as well as the nematic) by resonant small angle X\u2010ray scattering, TEM and 77Se NMR spectroscopy. The transition temperatures and associated enthalpies of four mixed polar/apolar bimesogens are given in Table\u2005TB phase are given in Figure\u200513 at 10\u2009\u00b0C\u2009min\u22121 is given in Figure\u2005Recently, we reported a compound  was taken to exclude the possibility of the NTB phase being a splay\u2010bend modulated structure. We observed that the small angle peak decreases from an average of 19.3\u2005\u00c5 in the nematic phase to 18.8\u2005\u00c5 in the NTB phase; this compares with a molecular length of 36.8\u2005\u00c5, obtained at the B3LYP/6\u201331G(d) level of DFT. The decrease in d\u2010spacing is comparable to that reported previously,TB phase.TB than in the nematic phase. Qualitatively, this would present as a reduced order parameter, however the lack of alignment in the NTB phase prevents us from giving quantitative values in this instance.We elected to study compound 9) spacers have been prepared and characterised, with all materials found to exhibit the twist\u2010bend nematic phase. The ability of a wide range of molecular structures to give rise to this mesophase represents a shift in our understanding of the twist\u2010bend phase from being a curious state of matter that is observed in a sparse number of materials to something universal that can be observed in a  large number of systems. A \u201cbent\u201d molecular structure is a prerequisite, with the thermal stability of the NTB phase showing a strong dependence on the intermesogen angle.R2=0.975) for these materials between the N\u2010I transition temperature and the NTB\u2010N transition temperature  are unlikely to yield an analogous linear relationship. These linear fits are only valid for materials with fairly rigid aromatic units and identical linking groups, thereby resulting in architectures that have three well\u2010defined sub\u2010units. Conversely, the ether\u2010linked, mixed ether\u2010alkyne or mixed ether\u2010methylene\u2010linked analogues of CB9CBWhen the linking unit is changed, this will inevitably lead to a change in the distribution of the conformers, and thus the average and also the distribution of the bend angles. As a consequence, the gross topology of the two molecules is no longer the same. Although a linear relationship exists for materials with comparable conformer distributions , in cases in which conformer distributions are significantly different , no linear relationship exists.TB phase is driven by gross topology, an argument that appears to be strongly supported by the present results, we speculate that this phase of matter should manifest on length scales beyond that of the molecule, in a manner analogous to the nematic and smectic phases exhibited by colloidal suspensions of virus particles.TB phase. These results and their relationship with macro\u2010 rather than nano\u2010systems suggest that the topological constraints, rather than the electrostatic interactions, of the molecules in the bulk phase are akin to viewing molecules as molecular grains of matter. Similarly, it is to be expected that supermolecular and supramolecular materials will eventually be found to exhibit the twist\u2010bend nematic phase, confirming that this state of matter is not confined solely to small molecules.If the incidence of the NTB to nematic transition temperature is important and shows that although the chemical make\u2010up of the mesogenic units drives the exact transition temperatures, the relationship between them apparently does not depend on the mesogenic units at all. We find that materials with different spacer lengths, linking groups and aspect ratios still exhibit this linear relationship, albeit with different slopes. The implication is that topology  and high\u2010resolution mass spectrometry. Purity was assayed by HPLC using both normal phase (SiO2) and reverse phase (C18\u2010SiO2) columns. Polarised optical microscopy (POM) was performed on a Zeiss Axioskop 40Pol microscope. Conoscopic figures were observed using an Olympus BH2 polarising microscope equipped with a Bertrand lens, an Olympus DPlan 100 PO 100x oil immersion objective with a numerical aperture of 1.25. An Olympus \u03bb\u2010waveplate (part no. 216958) was used to determine the sign of optical anisotropy. Images were captured using a Sony NEX 5R mirrorless digital camera (16.1 megapixels) affixed to the top of the microscope by a custom E\u2010mount to C\u2010mount plate. Temperature control during microscopy, both orthoscopic and conoscopic, was afforded by a Mettler FP82HT hotstage controlled by a Mettler FP90 central processor. Differential scanning calorimetry (DSC) was performed on a Mettler DSC822e calibrated before use against indium and zinc standards under an atmosphere of dry nitrogen. Small angle X\u2010ray scattering was performed on a Bruker D8 Discover using copper K\u03b1 radiation (\u03bb=0.15418\u2005nm) equipped with a temperature\u2010controlled, bored graphite rod furnace. Computational chemistry was performed at the B3LYP/6\u201331G(d) level of DFT in Gaussian G09.All materials were purified by column chromatography over silica gel, followed by filtration to remove insoluble matter and finally recrystallisation. Chemical structure was confirmed by NMR  should be addressed to the authors.SupplementaryClick here for additional data file."}
+{"text": "TB phase into an anticlinic smectic \u2018X\u2019 phase. Across all three series of compounds the length of terminal chain is seen to dictate, to some degree, the type of mesophase formed: shorter terminal chains favour nematic and NTB mesophases, whereas longer terminal aliphatic chains were found to promote smectic phases.Twelve symmetrical dimeric materials consisting of a nonamethylene (C9) spacer and either phenyl 4\u2010benzoate, phenyl 4\u2010benzoate or phenyl 4\u2010carboxylate mesogenic units were prepared and their mesogenic behaviour characterised by POM, DSC and XRD. All of the materials exhibited nematic phases with clearing points in excess of 200\u2009\u00b0C. Four compounds were found to exhibit the twist\u2010bend nematic phase, with one material exhibiting a transition from the N X or NTB,X/NTB phase is still hotly debated, with the heliconical \u2018twist\u2010bend\u2019 model proposed independently by Meyer2H\u2005NMR studies, measurement of the electroclinic effect, freeze\u2010fracture transmission electron microscopy (FFTEM) and carbon K\u2010edge SAXS.TB phases.In recent years there has been a resurgence of interest in liquid\u2010crystal dimers and bimesogens, driven by interest in wide temperature range blue phases,2H\u2005NMR experiment to show that a helix is not present in the NTB/NX phase.33 falling below zero, conversely, experimental studies have shown that this is not the case for CB7CB.TB/NX phases has provided a further challenge to future theoretical treatments.However, this view of the local structure has also been disputed, with a growing body of experimental evidence now interpreted to be counter the heliconical model. Specifically, Hoffmann used a novel TB phase, but also the molecular features that give rise to this unique phase of matter. Although predominantly exhibited by methylene\u2010linked bimesogens, the NTB phase has also been reported in bent\u2010coresystems,TB phase formation, with both imine,TB phase is known to exhibit local spontaneous chirality,There is significant interest not only in the local and bulk structures of the NTB phases and longer homologues (>C7) exhibiting anticlinic smectic C phases.Recently we demonstrated how the length of the terminal alkoxy chain in nonamethylene (C9)\u2010linked phenyl 4\u2010alkoxybenzoates dictates the mesophase behaviour of the system, with shorter homologues (<C7) exhibiting nematic and N4, 8 and 12 were prepared as described previously.Bis{1,9\u2010(4\u2010hydroxyphenyl)nonane} and compounds 1\u201312 were studied by a combination of polarised optical microscopy (POM) and differential scanning calorimetry (DSC). Phase identifications were made based on textures observed by POM, assisted where appropriate with small angle X\u2010ray scattering data (SAXS).The transition temperatures (\u00b0C) and enthalpies of transition (kJ\u2009mol\u22121) are given for each series of compounds in Tables\u2005The mesomorphic properties of compounds 1 and 2 both exhibit enantiotropic NTB phases, the latter material also exhibiting an additional smectic phase.TB phase is suppressed. The lower temperature \u2018SmX\u2019 phase exhibited by 2 is not seen, with a direct nematic to anticlinic smectic\u2005C transition being observed instead. A further increase in the terminal chain length from butyl to pentyl, shows that compound 4, affords a further increase in the onset temperature of the smectic CA phase with an additional monotropic\u2005B phase observed on cooling. Photomicrographs of the defect textures of the mesophases are presented in Figure\u2005Table\u20051 and 2 from the nematic to NTB phase yielded the typically broken stepped texture that is characteristic for this phase  to study the N\u2013SmCA mesophase exhibited by compounds 3 and 4 was measured by small\u2010angle X\u2010ray scattering. In both materials the layer spacing was found to be virtually temperature independent, with measured values of 23.3\u2005\u00c5 for 3 and 24.2 for 4. These values correspond to d/l ratios  level of DFT) of 0.55 for 3 and 0.53 for 4, indicating that the smectic CA phase in both materials is extensively intercalated. For compound 4, a temperature\u2010independent layer spacing of 24.3\u2005\u00c5 was observed in the B phase which leads to a d/l ratio of 0.54.The layer spacing of the smectic C3 and 4 exhibit a direct nematic to smectic CA phase transition without an intervening twist\u2010bend nematic phase we elected to construct phase diagrams . In the phase diagram between 4 and CB9CB, the NTB can be observed up to around 50\u2005wt\u2009% of 4, after which a direct nematic to smectic CA transition is observed. Linear fitting of the NTB\u2013N transition temperature as a function of concentration allows extrapolation of the virtual nematic to twist\u2010bend nematic transition temperatures for 3 and 4. This was determined to be 141.7 and 141.1\u2009\u00b0C for compounds 3 and 4, respectively. Notably, this virtual transition temperature occurs at a lower temperature than that of the N\u2013SmCA transition and hence is not observed in the pure materials. However, the virtual NTB\u2013N transition temperatures for 3 and 4 are still significantly higher than that measured for CB9CB (104.8\u2009\u00b0C).Binary mixtures between trans 4\u2010benzoate dimers. As with compounds 1\u20134, all materials exhibited enantiotropic nematic phases. The two homologues bearing shorter terminal chains  exhibited an additional twist\u2010bend nematic phase. Compounds\u20027 and 8 do not exhibit the NTB phase, instead a direct nematic to anticlinic smectic C phase was observed. Both the nematic and NTB phases can be identified from their optical textures, with photomicrographs presented in Figure\u2005Table\u20058). For compounds 7 and 8 an additional phase transition was observed by DSC at 96.2 and 114.6\u2009\u00b0C, respectively, which was classified as a B phase. Upon cooling from the anticlinic smectic C phase a transitory paramorphotic focal\u2010conic texture was observed  level of DFT).We next elected to study compound 8 the SmCA layer spacing, as determined by SAXS, was found to have a temperature\u2010independent value of 24.2\u2005\u00c5, corresponding to a d/l ratio of 0.55. Compounds 3, 4, 7, and 8 all exhibit SmCA phases with temperature\u2010independent d/l ratios in the region of 0.50\u20130.55, indicating that in all cases the smectic CA phase is extensively intercalated irrespective of terminal chain length or the type of mesogenic unit employed. The B phase exhibited by compounds 7 and 8 was also studied by SAXS, although the partial alignment present in the smectic CA phase of both materials was lost upon cooling into the B phase. The temperature independent layer spacing of the smectic B phase was measured to be 23.2 and 23.5 for 7 and 8, respectively, corresponding to d/l ratios of 0.52 for 7 and 0.54 for 8.For compound trans,trans4\u2010cyclohexylcarboxylate dimers are presented in Table\u2005TB phase, but instead show direct nematic to B phase transitions. Photomicrographs of the defect texture of the B phase exhibited by compound 9 are shown in Figure\u200512 has been confirmed previously by miscibility studies with 65OBC.The mesomorphic properties of four nonamethylene\u2010linked phenyl 9 and 10 results in the B phase exhibiting its natural textures, that is, homeotropic and mosaic, rather than the paramorphotic focal\u2010conic textures observed for the other compounds in this work. Compound 10 was studied by small\u2010angle X\u2010ray scattering , whereas compound 12 exhibits an additional smectic CA phase. Photomicrographs for the textures of the mesophases exhibited by 12 are given in Figure\u200511 and 12 were studied by SAXS. The layer spacing for the smectic B phase exhibited by compounds 11 and 12 was measured to be 23.64 and 24.68\u2005\u00c5, respectively, with both values found to be temperature independent. Using molecular lengths calculated at the B3LYP/6\u201331G(d) level of DFT (43.8\u2005\u00c5 for 11 and 44.5\u2005\u00c5 for 12) the measured layer spacings for 11 and 12 correspond to d/l ratios of 0.54 and 0.55, respectively, indicating that as with compounds 4 and 8 the B phase exhibited by methylene linked bimesogens is heavily intercalated.Compound 1\u20134) or phenyl cyclohexylbenzoate (5\u20138) mesogenic units exhibiting additional twist\u2010bend nematic or tilted smectic mesophases. The behaviour of compound 1\u20138 is in contrast to bimesogens incorporating phenyl bicyclohexylcarboxylate mesogenic units; compounds 9 and 10 exhibit nematic and B phases, with longer homologues also exhibiting additional SmA (11) or SmA and SmCA (12) phases. The ability to control the mesophases exhibited by a given bimesogen by chemically altering the mesogenic units is seemingly analogous to that of typical calamitic materials, where biphenyl benzoates will tend to give nematic and tilted phases and the analogous bicyclohexyl materials will preferentially exhibit higher\u2010order smectic phases.TB/NX phase and smectic mesophases are still rare, and as yet no theoretical treatments of such events have been provided.The present results show that in bimesogens the specific mesophases exhibited can be controlled by employing differing mesogenic units. All twelve dimeric materials exhibited a nematic mesophase, with those incorporating phenyl biphenylbenzoate  for nematic to NTB transition in compounds 1, 2, 5 and 6 are approximately two orders of magnitude lower than those for the nematic to isotropic transition in the same materials. There is a slight increase in the scaled transition temperatures (TNTB\u2013N/TN\u2013Iso) with increasing terminal chain length; however, further increases yield direct nematic to smectic phase transitions as discussed previously. The values of the associated enthalpies and entropies of the N\u2013I and NTB\u2013N transitions increase when the mesogenic unit is changed from phenyl biphenyl carboxylate (compounds 1 and 2) to a phenyl cyclohexylbenzoate (5 and 6), indicating that the choice of mesogenic unit not only impacts upon the specific thermal behaviour of a given material but also on its transitional properties. Comparison of the associated enthalpies and entropies of transition of 1, 2, 5 and 6 with those of CB11CBTB\u2013N transition the associated enthalpy and entropy of transition are significantly larger for CB11CB than for the compounds presented in this work. As the associated enthalpy (and thus entropy) of a transition are often taken to be indicative of the change in local structure at a phase transition, this discrepancy suggests that there is a bigger shift in mesophase structure at the NTB\u2013N transition of CB11CB than for 1, 2, 5 and 6. How this is borne out in terms of the properties of the NTB phase is presently unclear.As shown in Table\u2005TB phase becomes less stable in comparison to intercalated smectic phases as the proportionality of \u03c3\u2010 to \u03c0\u2010bonded structuring is increased. This is consistent with conventional calamitic low molar mass materials where smectic mesophases become stabilised with increasing chain length. However, it is also known that the presence of alicyclic rings systems also promotes nematic behaviour particularly when substituted with short aliphatic chains, but as the terminal substituent is extended their behaviour becomes more in keeping with long aliphatic chains. This will lead to stabilisation of smectic phase behaviour and that of the B phase in particular and thus is similar to the hydrophobic effect found for classical amphiphilic behaviour.We have demonstrated that the NAs a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re\u2010organized for online delivery, but are not copy\u2010edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file."}
+{"text": "The mechanisms underlying the protective effects involve Sirt1/FOXOs, Sirt1/NF-\u03baB, Sirt1/NOX, Sirt1/SOD, and Sirt1/eNOs pathways. In this review, we summarize the most recent reports in this field to recapitulate the potent mechanisms involving the protective role of Sirt1 in oxidative stress and to highlight the beneficial effects of Sirt1 on cardiovascular functions.The vascular endothelium is a layer of cells lining the inner surface of vessels, serving as a barrier that mediates microenvironment homeostasis. Deterioration of either the structure or function of endothelial cells (ECs) results in a variety of cardiovascular diseases. Previous studies have shown that reactive oxygen species (ROS) is a key factor that contributes to the impairment of ECs and the subsequent endothelial dysfunction. The longevity regulator Sirt1 is a NAD Endothehalation . TherefoAs a class III nicotinamide adenine dinucleotide- (NAD-) dependent histone deacetylase, Sirt1 has been demonstrated to regulate critical metabolic processes including oxidative stress, ageing, and apoptosis via deacetylation of a variety of substrates , 9. IntrIt is widely acknowledged that silencing information regulator complex (SIR complex) confers longevity for yeast . In yeasPolygonum cuspidatum. Our research team has validated that polydatin protects hepatocytes [So far, several natural and synthetic substances have been reported to activate Sirt1 and promote endothelial homeostasis. Resveratrol is the most widely used. Resveratrol is a subtype of phytoalexins that protects ECs against enhanced proinflammatory cytokines and reactive oxygen \u201317. Resvatocytes , small iatocytes , and kidatocytes  against atocytes . Moreoveatocytes . Quercetatocytes . In addiatocytes .By interacting with several target proteins, Sirt1 exerts a wide range of cellular functions including energy balance, lipid homeostasis, and especially, endothelial protection from vascular diseases . In agin+-dependent deacetylase, Sirt1 is capable of deacetylating numerous targets to protect ECs [\u03b1), nuclear respiratory factor 1 (NRF1), and eNOs [\u03baB p65 in the nucleus and decreased inflammation [\u03b1-induced CD40 elevation in human umbilical vein ECs (HUVECs) [Functioning as an NADtect ECs . By deactect ECs . Sirt1 ctect ECs . Downregtect ECs . Furtherand eNOs . Ghisaysammation . Increas(HUVECs) . It is a(HUVECs) . Autopha(HUVECs) , 40. Res(HUVECs) . Taken t2\u2212), hydrogen peroxide (H2O2), hydroxide (OH\u2212), and hypochlorite (OCl\u2212). which participate crucially in the impairment of ECs. ROS plays a pivotal role in many diseases including atherosclerosis, diabetic mellitus, and myocardial dysfunction [\u03baB, Sirt1/NOX, Sirt1/SOD, and Sirt1/eNOs pathways.Oxidative stress is characterized by the imbalance between the ROS production and oxidative stress resistance . ROS incfunction . There iFOXOs belong to a subgroup of Forkhead family of transcriptional factors. Invertebrates possess only one FOXO gene, whereas mammals have four FOXO genes: FOXO1, FOXO3, FOXO4, and FOXO6 . In respThe link between FOXO and Sirt1 indicates an evolutionarily mechanism for oxidative stress resistance. FOXO1, FOXO3a, and FOXO4 are indispensable for Sirt1-dependent cell survival against oxidative stress . It has In spite of the obscure interactions between Sirt1 and FOXO in response to oxidative stress, to a certain extent, their interaction with each other, indeed, benefits vascular ECs. Evidence showed that Sirt1-dependent activation of FOXO1 was crucial in vascular protection after the onset of oxidative stress . Consist\u03baB comprises of five subgroups including RelA or P65, RelB, c-Rel, p50/p105, and p52/p100, which remain quiescent because they are attached to an inhibitory protein I\u03baB. After IKK triggers I\u03baB phosphorylation and renders the degradation of I\u03baB, NF-\u03baB is activated and translocates from cytosol to the nucleus [\u03baB activation is closely related to reactive oxygen generation and greatly contributes to endothelial dysfunction. Several studies have revealed that antioxidant genes are under the control of NF-\u03baB. Through regulation of NF-\u03baB at the transcriptional level, a dramatic increase in mRNA levels for NF-\u03baB was detected and oxidative stress was induced after cadmium (Cd) exposure [\u03baB inhibitor SN50 dramatically reduces ROS production, indicating that oxidative stress is mediated by NF-\u03baB [The transcriptional factor NF- nucleus . NF-\u03baB aexposure . Moreoveby NF-\u03baB .\u03b1-induced injury through promoting Sirt1-induced repression of NF-\u03baB and ROS generation. The N-terminal domain of Sirt1 is a positive regulator of the endogenous Sirt1-dependent deacetylation capacity and promotes its physical association with NF-\u03baB p65 by deacetylating p65, thereby inhibiting NF-\u03baB transcription [\u03baB expression while depletion of Sirt1 via siRNA presented reduced resistance to ROS induced by hyperglycemia stress [\u03baB were attenuated in response to caloric restriction (CR) in cultured coronary arterial endothelial cells (CAECs). Intriguingly, sera obtained from CR animals showed antioxidant effects and NF-\u03baB inactivation. The aforementioned effect was Sirt1 dependent [\u03baB and Sirt1 could be involved in antagonistic crosstalk during the regulation of ROS in ECs. Sirt1 inhibits NF-\u03baB by directly deacetylating the p65 subunit or activating AMPK and PPAR\u03b1, which inhibits the NF-\u03baB pathway and naturally suppresses ROS generation. Additionally, NF-\u03baB transcription suppresses Sirt1 activation through ROS production [\u03baB signaling and controls ROS attack, and NF-\u03baB itself could decrease Sirt1 levels to increase ROS production.However, this process can be reversed through Sirt1 activation. Resveratrol protects HUVECs against TNF-cription , 37. In a stress . It has ependent . Furtheroduction . Taken tNADPH oxidase (NOX) family consists of several members including: NOX isoforms, two organizer subunits , two activator subunits , and two DUOX-specific maturation factors (DUOXA1 and DUOXA2) . The mem2\u2212 production [+-dependent. Decreases in NAD+ content induced by ROS tend to impair Sirt1 activity [+ content and Sirt1 levels to induce oxidized state in ECs. These effects were explained by the moderate and transient increase in ROS, which induced Sirt1 expression, inconsistent with the findings that short-term H2O2 treatment could enhance Sirt1 levels [Sirt1 participates in diminishing NOX production. It was confirmed that Sirt1 inhibition was engaged in upregulation of NOX oxidase subunits, p22phox, and NOX4, eventually leading to endothelial dysfunction due to Ooduction , 66. Meaoduction . It has activity . However1 levels , 69. Hen2O2 by binding to a superoxide anion with their metal zipper. Deficiency or impairment of metals in SODs including Cu-SOD, Zn-SOD, Ni-SOD, Mn-SOD, and Fe-SOD contributes to oxidative stress directly [SOD (superoxide dismutase), a group of metal-containing enzymes, is characterized by their ability to scavenge reactive oxygen species. SOD enzymes comprise of 3 members, including SOD1 (cytoplasmic), SOD2 , and SOD3 (extracellular). SODs have a pivotal role in oxidative stress resistance specifically in liberating Hdirectly .+ levels and Sirt1 expression was detected after nicotinamide mononucleotide (NMN) treatment in thoracic aorta. These effects were accompanied by MnSOD enhancement, which was probably modulated by Sirt1 to exert vascular antioxidant effect [Sirt1 has been shown to promote manganese superoxide dismutase (MnSOD) expression and increase the oxidative stress resistance in human retinal microvascular endothelial cell (RMECs). Sirt1 potentiates FOXO3a activation by deacetylating FOXO3a, which increases the transcription of downstream genes such as MnSOD. Therefore, Sirt1/FOXO/MnSOD may contribute to oxidative stress resistance in ECs . Moreovet effect . Rapidlyt effect . Similart effect . Therefo2\u2212 generation [2\u2212 generation. Therefore, oxidative stress could abolish eNOs activity through S-glutathionylation [NOs families comprise of three groups including neuronal nitric oxide synthase (nNOs) expressed in vascular smooth muscle, inducible nitric oxide synthase (iNOs) present in blood vessels under abnormal conditions, and endothelial nitric oxide synthase (eNOs) prominently expressed in ECs . eNOs maneration . The funnylation .2O2. Furthermore, Sirt1 has been shown to promote endothelium-dependent vasodilation by targeting eNOs for deacetylation. Sirt1 and eNOs colocalize in ECs, and subsequently, Sirt1 activates eNOs through deacetylating lysine 496 and 506, and as a result, NO production is increased. Consequently, its ability for antioxidant stress is enhanced because NO bioavailability is closely related to increased oxidative stress resistance [2O2-induced senescence in HUVECs, suggesting the phosphorylation of eNOs by Sirt1 is a promising strategy for combating premature senescence of HUVECs [The relationship between Sirt1 and eNOs in the process of defending oxidative stress has been reported extensively. It was demonstrated that Sirt1 agonist SRT1720 exerted salutary effects on expression of eNOs thus protecting HUVECs from senescence induced by Hsistance , 77. Consistance . In addif HUVECs . Intriguf HUVECs . In addif HUVECs . Therefo+. If NAD+ is overconsumed due to higher levels of Sirt1, mitochondrial biogenesis reduces and its stress resistance is reversed, suggesting that the beneficial effect of Sirt1 can only be achieved at low to moderate doses. Careful evaluation is needed to determine therapeutic doses in clinical practice [Although Sirt1 may be considered to be an optimal therapeutic target for vascular diseases, discreet evaluations should be conducted regarding the dosage to be used for therapy. As reported, 2.5\u20137.5-fold overexpression of Sirt1 prevents heart from oxidative stress via Sirt1/FOXO axis accompanied by the consumption of NADpractice .\u03baB, Sirt1/NOX, Sirt1/SOD, and Sirt1/eNOs. Among these factors, eNOs and SOD are downstream of FOXO, which could also activate Sirt1 and trigger the signaling pathway through positive feedback. Therefore, the positive network of the several antioxidant molecules would amplify the effect to defend oxidative stress. However, Sirt1 also serves as a key target of NF-\u03baB, whose transcription suppresses Sirt1 activation, resulting in the increase in ROS generation and a decrease in ROS scavenging. Furthermore, antagonist effect exists between SOD and NF-\u03baB. Transactivator of transcription (Tat)-SOD protein can hinder NF-\u03baB activation, defending the oxidation-driven atherosclerosis in HUVECs [\u03baB and reverse monolayer hyperpermeability induced by release of hemoglobin with hemolysis [\u03baB was suppressed by NOX inhibition in ECs, indicating the interplay between NF-\u03baB and NOX [\u03baB signaling was also shown to engage in aggravated endothelial dysfunction due to high-dose intravenous iron supplementation [ROS causes progressive deterioration of the structure and function of ECs. As an antioxidative stress molecule, Sirt1 has been identified and studied to determine its role in ROS resistance in ECs. Intriguingly, transient increase of ROS could induce Sirt1 which, in turn, causes a decrease in ROS. However, high ROS levels diminish Sirt1 activation. The explanation for these effects may involve different cellular activities to counteract ROS. There is a complex signaling network for Sirt1-mediated ROS reduction involving Sirt1/FOXOs, Sirt1/NF-n HUVECs . Togetheemolysis . Activat and NOX . The NOXentation . It has entation . ProbablDespite the promising evidence, however, due to the fact that excessive expression of Sirt1 seems to exert the opposite effect, appropriate upregulation of Sirt1 may be taken into consideration for vascular disease therapy. Therefore, much more studies in high quality should be carried out for Sirt1 elevation regarding the optimal dosage as therapeutic use and its practical potential for disease prevention and protection in clinical application."}
+{"text": "There are seven proteins (SIRT1-7) in the human sirtuin family, among which SIRT1 is the most conserved and characterized. SIRT1 in the brain, in particular, within the hypothalamus, plays crucial roles in regulating systemic energy homeostasis and circadian rhythm. Apart from this, SIRT1 has also been found to mediate beneficial effects in neurological diseases. In this review, we will first summarize how SIRT1 in the brain relates to obesity, type 2 diabetes, and circadian synchronization, and then we discuss the neuroprotective roles of brain SIRT1 in the context of cerebral ischemia and neurodegenerative disorders.Sirtuins are evolutionarily conserved proteins that use nicotinamide adenine dinucleotide (NAD Additionally, some sirtuins have been reported to exhibit demyristoylase (SIRT2), ADP-ribosyltransferase (SIRT4 and SIRT6), and demanlonylase and desuccinylase (SIRT5) activities (Sirtuins are homologs of yeast silent information regulator 2 (Sir2). Sir2 has attracted the attention of researchers given its involvement in longevity . The mam+ dependent deacetylase that removes the acetyl groups from protein substrates to add to the ADP-ribose, a product from the cleavage of NAD+. NAD+ is a dinucleotide with one nucleotide contains an adenine and the other contains nicotinamide   is an inner mitochondrial membrane protein that can uncouple oxidative phosphorylation from respiration/ATP production. This is done via dissipation of the proton gradient, in which protons are returned to the mitochondrial matrix. UCP2 is found in many different tissues, including the brain, and has been shown to be involved in energy balance , homeostPeroxisome proliferator-activated receptor \u03b3 (PPAR\u03b3) coactivator-1\u03b1 (PGC-1\u03b1) is a transcriptional coactivator and major regulator of mitochondrial biogenesis and several metabolic processes . StudiesNRF-1, specifically, can regulate the activation of the nuclear-encoded mitochondrial transcription factor A (TFAM), which can bind to mtDNA and stimulate mitochondrial DNA replication and increase the expression of mitochondrial genes . As a reThe increase of mitochondrial gene expression also stimulates several metabolic processes depending on the tissue type , 36. In Maintaining quality mitochondrial pools is essential for cell health and viability. Mitochondrial components are typically damaged by the accumulation of ROS\u2014a byproduct of the mitochondrial electron transport chain\u2014which typically occurs during conditions of stress. Due to its close proximity, ROS overproduction damages mtDNA that may ultimately contribute to neurodegenerative disorders, stroke, cancer, and age-related diseases \u201342. ConsSeveral studies have shown that SIRT1 interacts with components of the autophagy machinery including Forkhead box O3 (FOXO3)\u2014a transcription factor heavily associated with autophagy induction \u201347. In tThe hypothalamus is the control center for homeostasis. In the arcuate nucleus of the hypothalamus, the proopiomelanocortin (POMC) neurons suppress appetite while the activation of agouti-related peptide/neuropeptide Y (AgRP/NPY) neurons stimulate appetite . The venSIRT1 in the POMC neurons is required to protect against high calorie-induced obesity. When challenged with a hypercaloric diet, POMC-SIRT1 mutant mice showed reduced energy expenditure and increased body weight . InteresAnother mechanism in which SIRT1 modulates systemic homeostasis is through deacetylation of Forkhead box protein O1 (FoxO1). FoxO1 is a downstream transcription factor in the insulin signaling pathway. Hypothalamic FoxO1 activation or overexpression inhibits the anorexigenic effects of insulin , increas+ levels in the hypothalamus, suggesting that the metabolic status could influence the function of hypothalamic SIRT1  and activates the signal transducer and activator of transcription 3 (STAT3), which further regulates gene expressions to affect energy homeostasis. The leptin-induced protective mechanisms against obesity are dependent on SIRT1 in the POMC neurons. In POMC-SIRT1 deficient mice, leptin-mediated activation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling pathway and the suppression of food intake were disrupted . In addiic SIRT1 . In suppic SIRT1 . In addiic SIRT1 . In contic SIRT1 .In the aforementioned studies, the activation of hypothalamic SIRT1 negatively regulated energy balance and protected against obesity. Contrary to these findings, studies also reported a positive energy regulation by brain SIRT1. Apart from leptin, ghrelin is another peptide released peripherally and acts on the central nervous system (CNS) to regulate metabolism. Ghrelin is produced by the stomach and activates AMPK in the hypothalamus to increase appetite. In rodents, the intracerebroventricular (ICV) infusion of the SIRT1 inhibitor, EX-527, blunted the ghrelin-induced food intake, thus demonstrating that ghrelin is dependent on hypothalamic SIRT1 to stimulate appetite , 59. SimObesity is a leading risk factor for type 2 diabetes (T2DM). The accumulation of fat, especially visceral fat, progressively enhances insulin resistance and eventually leads to T2DM , 63. GivResveratrol is a potent SIRT1 activator that improves glucose homeostasis. Studies demonstrate that brain SIRT1 at least partially contributes to the resveratrol-mediated glucose balance . ICV infin vitro and in vivo, whereas resveratrol treatment increased the glycolysis rate in primary neurons  and the insulin signaling pathway. In this case, central PI3K signaling was enhanced in neuronal SIRT1 deficient mice . Another neurons . It is r neurons . For exa neurons .The circadian rhythm is a 24 h endogenous cycle that allows organisms to synchronize their physiology and behavior to the daily cycle of daylight and darkness , 70. TheThe molecular mechanism of the circadian rhythm consists of a set of transcriptional activators and repressors involved in positive and negative autoregulatory feedback loops , 71. In + levels regulated by its rate-limiting enzyme NAMPT, which is positively regulated by the core clock genes CLOCK-BMAL1  mice, a model of Alzheimer's disease, exhibit disruptions in the circadian locomotor activity under dim light and constant darkness along with impairments in re-entrainment to phase change schedules  . In a siThe neuroprotection against cerebral ischemia by SIRT1 is achieved through multiple mechanisms. Following ischemia, stressors, such as DNA damage and oxidative stress activate the tumor suppressor gene, p53, which mediates apoptosis . IschemiSIRT1 dependent endothelial nitric oxide synthase (eNOS) modulation is another beneficial mechanism. Nitric oxide (NO) is a vasodilatory factor that is produced by endothelial nitric oxide synthase (eNOS) in endothelial cells. Acetylated eNOS was significantly increased at 2 h after BCAS in wild-type mice, whereas in SIRT1-Tg mice, the acetylation of eNOS was not observed . IncreasAs briefly mentioned above, SIRT1 can also mediate protection by retaining the integrity of white matter . White mImportantly, Sirt1 is required in the neuroprotection elicited by ischemic preconditioning (IPC) or resveratrol preconditioning (RPC) , 67. IPCIn addition to providing neuroprotection against cerebral ischemia, the activation of SIRT1 has been shown to confer protection against neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD) , 100. ThThe benefits of SIRT1 in neurodegenerative diseases was first reported by Graff et al. in a CR model . Graff eAD is a neurodegenerative disease that can be either early-onset or late-onset. Early-onset is associated with a genetic contribution to the disease's etiology, while the late onset etiology is more complicated and likely multifactorial . In the Recent evidence has linked SIRT1 activity with the interference of the factors and-or processes that produce NSP's and NFT's Figure . CR in mIn an oxidative stress model of neuroblastoma SK-N-BE cells, the SIRT1 activator resveratrol was administered to determine its effect on neurodegenerative oxidative stress and protein aggregation . ResveraSNCA gene which produces the protein in a misfolded state  in the substantia nigra pars compacta (SNpc) . The losed state . Anothered state . OverallOnce again, SIRT1 may play a protective role in neurodegenerative disorders, PD included. There is evidence that suggests there may be genetic correlations between SIRT1 and PD, SIRT1-activated anti-PD signaling, and SIRT1-dependent neuroprotection in various models. Extracellular \u03b1-synuclein accumulation leads to mitochondrial dysfunction and a reduction of SIRT1 expression . The dowin vitro human DA neuronal culture conferred resilience when the cultures were subjected to oxidative stress (There is evidence that shows that cellular signaling resultant from SIRT1 activation, or pathways that include SIRT1, are involved in the reduction of \u03b1-synuclein and promotion of DA neuron survival Figure . The appe stress . This ree stress . Ultimate stress . In thise stress . A studye stress . Again, e stress . In a PCe stress . It is cInflammation within the CNS can exacerbate or potentially initiate PD pathology . The celThe removal of \u03b1-synuclein in a healthy DA neuron entails the use of a few cellular clearance mechanisms, most of which involve some type of autophagy . One of Considering the multitude of evidence supporting SIRT1's neuroprotective potential against PD and the dynamic range of mechanisms in which that protection is enacted, SIRT1 appears to be an optimal target for the therapeutic treatment of PD. However, recent attempts to enhance SIRT1 expression or activity directly, not through activators, has not shown the same robust results. A study utilized a CNS SIRT1 overexpression mouse model to study the MPTP model of PD. As compared to controls the SIRT1 overexpression mouse did not confer protection against acute toxicity of MPTP in nigrostriatal DA neurons . AdditioAnother neurodegenerative disease in which SIRT1 has been investigated is HD. HD is a genetically autosomal dominant disease in which the HD gene produces a mutant version of the protein. Extended CAG-repeats in the HD gene results in the translated protein acquiring a pathological conformation, affecting its solubility and promoting aggregations . AggregaLike other neurodegenerative diseases, the role of SIRT1 has been investigated in the context of HD pathology. In the R6/2 mouse model of HD, the levels of metabolic and cell cycle regulators were assessed. SIRT1 mRNA and protein were increased in this model but this increase did not correlate to increased activity as shown by no significant change in p53 acetylation . This chMany studies have intentionally augmented SIRT1 in the contexts of HD to elucidate whether it is part of the pathology or potentially therapeutic. In an HD mouse model, SIRT1 was overexpressed, improving motor functions and pathological metabolic functioning . SIRT1 oThe role of SIRT1 in HD certainly warrants further investigation. Though there are pathological increases in SIRT1 in neurons suffering from HD pathology, this may be a compensatory mechanism due to the inhibition of SIRT1 by the mutant HD protein. In studies overexpressing SIRT1, HD pathology has been ameliorated and this protection is dependent on SIRT1's activity. Taken together, these lines of evidence suggest that SIRT1 is inhibited in HD pathology and there may be an increase in its expression for compensatory reasons. Furthermore, the loss of SIRT1 deacetylation activity may contribute to HD pathology and restoration of SIRT1 activity likely possess therapeutic potential against the disease.In summary, any dysregulation of brain SIRT1 activity can have devastating consequences in terms of metabolism, circadian synchronization, and neurological function. Given that SIRT1 is highly specialized distributed in the hypothalamic nuclei, it is no surprise that brain SIRT1 is a major contributor to the systemic network of metabolic homeostasis. It should be noted that, nowadays, accumulated evidence supports a reciprocal relationship between brain and peripheral tissues in metabolic benefits, circadian oscillations and neurological functions , 134. AlAlthough most studies discussed above have used resveratrol as the SIRT1 activator, limitations remain for the resveratrol-induced SIRT1 activation. One study reported the activation of AMPK by high-dose resveratrol, suggesting the indirect effects of SIRT1 through AMPK pathway as well as the off-target effects of resveratrol . As a po+ pathway. As mentioned before, NAD+ is the rate-limiting co-substrate for SIRT1. Thus, increased NAD+ levels is presumably to activate SIRT1. So far, to supplement NAD+ precursor and boost NAD biosynthesis has been the main approaches to alter NAD+ levels. Experimental studies have reported beneficial effects of NAD+ precursor supplementation (+ precursor, effectively increased NAD+ levels in elders (+ or NAD+-induced SIRT1 activation. Therefore, pharmacological agents that are ligand-specific and tissue-specific are warranted to further clarify the functions of SIRT1 in biological and pathological events.In addition to resveratrol, another promising target to activate SIRT1 is the NADentation \u2013140. Curn elders . Again, JX, CJ, NK, IE, and MP-P cooperated to write the article and revised the content. MP-P and JX outlined the manuscript. MP-P supervised the work.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."}
+{"text": "Dear Editor,With an estimated 240 million chronically infected people worldwide, hepatitis B virus (HBV) infection is a major public health problem  were treated with the PI3KC3 inhibitor 3-methyladenine (3-MA), the Rab7 inhibitor CID1067700 (CID) or the lysosome inhibitor chloroquine (CQ). Immunofluorescence microscopy showed that the number of LC3 puncta was decreased by 3-MA treatment and increased following treatment with CID and CQ. Moreover, the ratios of LC3II/Actin were applied for assessing the autophagic activity, with beta-actin used as the internal reference for normalization , suggesting that increased HBsAg production after Rab7 silencing is not due to transcriptional regulation. Moreover, there was also no effect on HBV promoter activity by silencing Rab7 .Taken together, we examined how HBV replication was modulated by late phase of autophagy in the present study. While autophagy initiation was important for efficient HBV replication, a complete autophagic process led to the degradation of a significant part of HBV virions and HBsAg .Yong Lin, Chunchen Wu, Xueyu Wang, Thekla Kemper, Anthony Squire, Matthias Gunzer, Jiming Zhang, Xinwen Chen and Mengji Lu declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by the any of the authors.Supplementary material 1 (PDF 573 kb)Below is the link to the electronic supplementary material."}
+{"text": "Here, we describe the structure and function of the ALDH from Thermus thermophilus (ALDHTt) which exhibits non-canonical features of both dimeric and tetrameric ALDH and a previously uncharacterized C-terminal arm extension forming novel interactions with the N-terminus in the quaternary structure. This unusual tail also interacts closely with the substrate entry tunnel in each monomer providing further mechanistic detail for the recent discovery of tail-mediated activity regulation in ALDH. However, due to the novel distal extension of the tail of ALDHTt and stabilizing termini-interactions, the current model of tail-mediated substrate access is not apparent in ALDHTt. The discovery of such a long tail in a deeply and early branching phylum such as Deinococcus-Thermus indicates that ALDHTt may be an ancestral or primordial metabolic model of study. This structure provides invaluable evidence of how metabolic regulation has evolved and provides a link to early enzyme regulatory adaptations.Aldehyde dehydrogenases (ALDH) form a superfamily of dimeric or tetrameric enzymes that catalyze the oxidation of a broad range of aldehydes into their corresponding carboxylic acids with the concomitant reduction of the cofactor NAD(P) into NAD(P)H. Despite their varied polypeptide chain length and oligomerisation states, ALDHs possess a conserved architecture of three domains: the catalytic domain, NAD(P) The primary role of ALDH in humans and other mammals is the protection of the body from toxic compounds. For example, ALDH from liver mitochondria metabolizes acetaldehyde , the oxidation product of ethanol by alcohol dehydrogenase, to acetic acid. In half of the Asian population, a single amino acid residue change inactivates the liver mitochondrial ALDH, causing severe alcohol intolerance. The drug disulfiram (Antabuse), which is used in the treatment of alcoholism, inhibits the liver mitochondrial ALDH3. In addition to its detoxification role, ALDH is also important for the biosynthesis of retinoic acid4 and GABA neurotransmitter metabolism5. In recent times ALDH have also become a prime target in cancer research due to the abnormal activity of human ALDH in cancer disease models6. Interestingly, this group of enzymes has a role in non-enzymatic conditions, such as osmotic stress reduction7 and mammalian cornea UV exposure protection8.Found in both prokaryotes and eukaryotes, aldehyde dehydrogenases (ALDH) (EC 1.2.1.3) constitute a large family of NAD(P)-dependent enzymes with molecular weights of 50\u201360\u2009kDa. The human genome encodes for 19 known ALDHs9. Nonetheless, the range of possible substrates and their corresponding affinity in prokaryotic enzymes preclude a similar classification.Enzymatically, mammalian ALDHs can broadly be classified as cytosolic (Class 1), mitochondrial (Class 2) and those expressed in tumor, stomach and cornea (Class 3)Escherichia coli10. This is primarily due to their respective multiple roles with non-redundant independent regulation. Interestingly, unlike many other systems, ALDH studies often originated in human enzymes rather than bacterial ones, leading to rather inaccurate descriptions of \u2018atypical\u2019 features. For example, the metabolically-specific lactaldehyde and phenylacetalaldehyde dehydrogenases from E. coli were only characterized robustly after the human ALDH1/2/3, and were found to have differing cofactor specificities, rate-limiting step and salt bridge tendencies10. Considering that there are ~ 300 different domain architectures within the ALDH-like clan (Pfam: CL0099), the substrate and cofactor specificity are far from easily predicted computationally. This is further complicated by different oligomerization states.Despite the availability of various ALDH superfamily structures (>140 released and >20 unreleased structures), much of the attention has been on mesophilic enzymes including those of human and the archetypal Tt. Glu268 acts as the general base necessary for Cys302 activation through deprotonation in both the dehydrogenase and esterase reactions11. This reaction consists of (i) activation of the catalytic Cys302 by a water-mediated proton abstraction by Glu268, (ii) a nucleophilic attack by the Cys302 thiolate group on the electrophilic aldehyde, (iii) formation of the tetrahedral thiohemiacetal intermediate  with concomitant hydride transfer to the NAD(P)+ pyridine ring, (iv) hydrolysis of the thioester intermediate from the previous step and finally (v) the dissociation of the reduced cofactor [NAD(P)H] and subsequent enzyme regeneration by NAD(P)+ binding. Steps (i) and (iv) very likely require a Glu268-bound water molecule to facilitate Cys302 deprotonation and the subsequent hydrolysis of the thioester intermediate2.Through site-directed mutagenesis experiments, it is known that the catalysis occurs as a five-step reaction mediated by three highly-conserved residues Cys302, Lys192 and Glu268 (human ALDH2 numbering), which corresponds to Cys295, Lys182 and Glu261 in ALDHGeobacillus thermodenitrificans14, Pyrobaculum sp. (PDB ID: 4H73 and 4NMJ) (no primary citation available), Geobacillus thermoglucosidasius (PDB ID: 5J78)15 and the robustly characterized glyceraldehyde-3-phosphate dehydrogenases from Trichomonas tenax16 and Sulfolobus solfataricus17, and the presence of native ALDHTt as a contaminant during T. thermophilus caa3-cytochrome oxidase crystallization18, motivated us to investigate ALDHTt biochemically and structurally.Recent studies on interesting ALDHs from (hyper) thermophilic bacteria and archaea, including Tt and truncated mutants thereof. In contrast to other thermophilic ALDHs, but in agreement with mesophilic enzymes, the enzymatic characterization suggests the ability of ALDHTt to utilize both NAD+ and NADP+ as a cofactor. It also exhibits a broad range of substrate specificity. ALDHTt contains an unusually extended C-terminal arm forming novel interactions with the N-terminus in the quaternary structure which interacts closely with the substrate entry tunnel. The presence of a full complement of both N- and C-terminal residues in ALDHTt, in a deeply branching bacterial lineage such as Thermus, gives a structural snapshot of the early evolution of ALDH. This lends detail as to why terminal truncations were favored in evolutionarily more advanced ALDHs.In this article, we report the crystal structure of native ALDHTt, two copies related by a non-crystallographic symmetry (NCS) were present in the asymmetric unit. Based on PISA analysis19, however, it was clear that the tetramer assembly in ALDHTt consists of a combination of both NCS and crystallographic symmetry perpendicular to each other, forming a 222 symmetry , although the higher \u0394Gint for tetrameric assembly shifts the thermal equilibrium away from a dimeric assembly.The biologically active form of ALDH is a homotetramer. In the case of ALDHtry Fig.\u00a0. An inte2 out of a total of 22,900\u2009\u00c52, created by a network of 32 hydrogen bonds and 13 salt bridges. The A-C interface exhibits a slightly weaker interaction with a BSA of 2,770\u2009\u00c52 .PISA interface analysis shows strong interactions between the A-B dimer, with a buried surface area (BSA) of 3,500\u2009\u00c5The most striking feature of the tetramer is the orientation of the C-terminal arm extension in the overall quaternary structure. It wraps around the outside of the symmetry related dimer pair  comprising a Rossman fold, (ii) the catalytic domain (267\u2013458) and (iii) the oligomerization domain (126\u2013147\u2009+\u2009494\u2013501).  are separated by two loops, a short linker loop region containing Glu261 (261\u2013267), required to activate the catalytic cysteine Cys295, and a long inter-domain linker (459\u2013493) harboring the aldehyde anchor loop (464\u2013466). This anchor loop, which has been previously shown to contain regulatory residues such as the substrate entry channel (SEC) mouth residue20 and the gating aromatic residue21, interacts with the substrate and product. The catalytic and cofactor-binding domains form a central tunnel through the monomer with NAD(P)+ at one side and the classical entrance for substrate on the opposite side. The catalytic residues are deep within the center of the tunnel ~16\u2009\u00c5 from the cofactor binding site to Glu261 and substrate entry tunnel to the catalytic Cys295. The tunnel is ~5\u2009\u00c5 in diameter at its widest point.The ALDHDHs Fig.\u00a0: (i) TheTt515 was solved with NADP+ bound in the expected cofactor binding cleft of the monomeric subunit. As previously observed with other ALDH structures24, the electron density of the nicotinamide moiety was weak compared to the ADP-ribose part of the cofactor. Similarly to the human mitochondrial ALDH and ALDH1A213, the NADP+ cofactor is observed in the extended conformation in our structure. Comparison of the NADP-bound with apo structures did not reveal significant conformational changes for the adenosine pyrophosphate accommodation within the cofactor binding cleft. In the binding site the adenosine ribose interacts with Lys182 and Thr156. The 2\u2032-phosphate group is positioned in the ceiling of the binding cleft with O2 and O3 within 3\u2009\u00c5 distance from Glu185 carboxyl group. While these unfavorable contacts could result in electrostatic repulsion, the 2\u2032 phosphate is stabilized by hydrogen bonds with the Glu185 backbone amide group in addition to Ser184 and Lys182 side chains. The pyrophosphate moiety is hydrogen bonded to the backbone amide group of Ser240, its side chain and to the Arg342 sidechain. Finally, the nicotinamide ribose has O2 and O3 hydrogen bonded with Glu404, while its carboxamide nitrogen is hydrogen bonded to Leu262 ing Fig.\u00a0. As prev295 Fig.\u00a0 to act a261 Fig.\u00a0. In the Besides the catalytic residues orientation upon cofactor binding, another part of the structure seems to undergo subtle movements during catalysis. Indeed, our crystal structures show differences in the orientation of Leu262 and Gly263 backbone amide and carbonyl groups. In the apo structures, the \u201cIn\u201d orientation of Glu261 is associated with a hydrogen bonding between one of the Glu261 oxygens and the amide group of Gly263 Fig.\u00a0. This inThe recombinant wild type crystals yielded a structure with a product molecule, propanoic acid, in monomer A and B. The product is ~6\u2009\u00c5 from the catalytic thiol, which is in the attacking conformation, while the Glu261 can be observed in the \u201cIn\u201d conformation in monomer B. Interestingly, the ligand, situated inside the aldehyde anchor loop within hydrogen bonding distance to Arg294, Thr296, Gly464 and Ala465, is observed in two different orientations Fig.\u00a0. In monoTt starts after the oligomerization domain and terminates in a 3-turn helix, which interacts with the N-terminal residues of its opposing monomer (A-D). The novel orientation of the tail contributes to salt bridge formation and hydrogen bonds between monomers A/B and A/D. This is governed by three pivot or fulcrum points on the tail  interacting together via a hydrogen bond. In ALDHTt an additional salt bridge between Gln507 and Glu501 further\u00a0stabilizes the \u201cNotch\u201d region. The current ALDHTt structures do not indicate that this movement is possible due to the increased length and differing orientation. The tail does not make the distinctive \u201cL\u201d shape reported for ALDH7A1. It rather deviates again in the \u201cHook\u201d region creating a straight line path from oligomerisation domain to the N-terminus of a neighboring monomer. The \u201cCrook\u201d of the tail (Leu508-Gln510) is conserved in both structures as well as the interacting anchor loop residues (Gly464-Glu466). This conservation is thought to be a defining characteristic of family 7 of the ALDH superfamily, so it is possible that ALDHTt should be classified in family 7. In all ALDHTt structures, where the tail is persistently closed, the anchor loop is also closed. The ALDH7A1 structure terminates four residues after the \u201cCrook\u201d allowing room for the tail to fold into a hydrophobic pocket in the side of the catalytic domain. However, the ALDHTt terminates twenty residues after the \u201cCrook\u201d leaving insufficient space for a long tail to fold away from the substrate entrance tunnel. This swinging of the tail into an open position is unlikely in ALDHTt, due to Lys105 pointing up into the interdomain cleft , strengthening them and making it far stronger than mesophilic counterparts. The linchpin of these interactions revolves around an additional salt bridge in the oligomerisation domain of each monomer, Arg126-Glu136. The Arg126 of monomer D points toward the pivotal tail fulcrums and creates a central interaction point between the \u201cCrook\u201d and \u201cHook\u201d on the tail of monomer A, the anchor loop of monomer B and the oligomerisation domain of monomer D. The Arg126-Glu136 is thought to be the strongest of salt bridgesTt515 which removes all residues distal to the \u201cCrook & Hook\u201d and (ii) ALDHTt508, a mutant which removes the \u201cCrook & Hook\u201d entirely but retains the \u201cNotch\u201d to try and model other tail containing ALDHs. It was expected that at least one of the truncated mutants of ALDHTt could produce an open conformation of the protein and obtain substrate/product bound structures. However, ALDHTt mutants could only be crystalized in a closed and compact conformation. ALDHTt515 demonstrates no movement in the anchor loop or salt bridge interactions, leading us to believe that the last 15 residues of the protein play a minor role in the active site regulation. The ALDHTt508 structure is \u201copen\u201d as the \u201cCrook\u201d of the tail has been removed, but there is still no movement in the anchor loop to an open position. It is worthy to note that the electron density maps were of a reduced quality in the C-terminal region of the truncated mutants compared to the native or the full length recombinant proteins, highlighting the role of the extended C-terminal tail in stabilizing the overall structure.To probe the significance of the tail and its fulcrum points, two truncated tail mutants were made: (i) ALDHT. thermophilus HB8 strain is the only one with a significantly shorter tail within its genus (515 vs. 530 residues). Within the Deinococcus-Thermus phylum, however, not all are thermophilic bacteria. A careful DELTA-BLAST search against all structures in the PDB indicated the absence of a similar long C-terminal tail. Nonetheless, this tail is present not only within the Deinococcus-Thermus phylum , Pyrinomonas methylaliphatogenes  and Thermaerobacter marianensis . Despite a similar extensive search using only the C-terminal tail sequence (EYSGRLQLAQMDTGYVSPKAPTPWGEVLGL) against protein sequences outside the Deinococcus-Thermusphylum, only a partial sequence from Kouleothrix aurantiaca  was identified as containing the fragment SGKLQLAQMDTDYIAPKSP.Comparison with homologous sequences Table\u00a0 shows thn-octyl-\u03b2-glucoside (BOG) molecules originating from the detergent used for caa3-oxidase purification;  are found deep within the centre of the tetramer. They lie in a highly positive pore created by the oligomerisation domain and inter-domain linkers of each monomer. Pores and tunnels also exist between monomer interfaces in the tetrameric assembly and these interfaces are highly positively charged in the ALDHTt structure, far more so than many other known ALDH structures . Further assays were performed at both mesophilic temperature (25\u2009\u00b0C) and elevated thermophilic-like temperature (50\u2009\u00b0C). Although T. thermophilus grows best at 70\u2009\u00b0C, a lower temperature was used in the assays to minimize evaporation of certain volatile substrates and maintain stable readings.As the cofactor used in any enzyme assay has a significant effect on its readout, we first determined the preferential cofactor using a set of substrates for the recombinant wild type enzyme ALDHTt508 and ALDHTt515 were slightly more active , perhaps due to the presence of small amounts of ammonium sulfate acting as a stabilizing agent. Attempts to assess enzymatic activities at the higher temperature of 85\u2009\u00b0C were not successful as both mutants rapidly\u00a0lost their function compared to ALDHTt530 where it was still active up to 4\u2009min, consistent with\u00a0the Tm of ALDHTt530 of 84\u2009\u00b0C. Taken together, the relative stability of the recombinant enzymes was in the order ALDHTt530\u2009>\u2009ALDHTt515\u2009\u2248\u2009ALDHTt508 + binding sites do not belong to a single common family as classified in32 with ALDHTt situated in Group III_NADP family. Due to the poor discriminatory power of this family, it is not possible to state whether the enzyme binds to NAD+ and/or NADP+ on the basis of sequence alone. Despite the co-crystals of the truncated mutant ALDHTt515-NADP+ being significantly larger and better diffracting than those with NAD+, enzyme kinetics data described in this work indicates that the preferential cofactor is NAD+. In addition, these data are supported by the observed proximity of the 2\u2032 phosphate to Glu185, which gives the structural basis for NAD+ preference in ALDHTt.ALDHs are NAD(P)Tt with NADP+ shows no major conformational changes between the apo and cofactor-bound structures. The effect of NADP+ binding is observed locally on the catalytic residues Glu261 and Cys295 side chains which adopt classical orientations observed with cofactor bounded ALDH25. It is worthy to note here that the electron density of the nicotinamide moiety was weaker than the rest of the cofactor. This is mainly due to the high flexibility of this part of the cofactor, thought to sample different conformations during catalysis35.The structure of ALDHt-FDH, are believed to play a role in the cofactor stabilization26. Consequently, these movements are likely to play a role during catalysis, as they involve direct interactions with the general base Glu261 and the nicotinamide moiety of the cofactor. As for ALDH2, the proximity of Glu261 with Cys295 argues for a direct activation of the catalytic cysteine by the glutamic acid. The latter, being in the \u201cIn\u201d conformation, is stabilized by a hydrogen bond with Gly263. Upon cofactor binding, Glu261 rolls back to make a place for the cofactor, this movement is accompanied by a local 180\u00b0 flip of the main chain linker loop to bring Leu262 carbonyl group toward the carboxamide nitrogen of the nicotinamide for stabilization. This will allow for the hybride transfer to occur, and for another 180 \u00b0 flip of the main chain linker loop along with \u201cIn\u201d positioning of Glu261 following cofactor exit. This last step is supported by our product-bound structure where the \u201cIn\u201d conformation of Glu261 is observed. Indeed, in the full-length recombinant protein Glu261 is observed in the \u201cIn\u201d conformation, which may be indicative of an intermediate state depicting the molecule leaving the tunnel after catalysis, with repositioning of the glutamic acid. The different orientations of the product in monomers A and B is likely to provide snapshots of product handling throughout the aldehyde anchor loop. It is worthy to note that a similar product orientation was previously observed with ALDH1A336.Besides moving Glu261 away from the \u201cIn\u201d position, cofactor binding induces more subtle changes in the active site. This concerns a 180\u00b0 main chain flip around residues of the linker loop Leu262 and Gly263 which interact with Glu261 and the nicotinamide moiety of the cofactor, respectively. These\u00a0kinds of interactions between the linker loop and the nicotinamide moiety, as previously reported for CTt, we noticed the presence of an extended C-terminal tail. This is not surprising as similar extensions have been seen in the ba3-oxidase, caa3-oxidase and cytochrome c552 of T. thermophilus previously. In contrast, thermophilic targets are expected to have shorter loops and tighter packing39.Initially, when we solved the structure of the native ALDH40, with the exception of the human tetrameric ALDH7A130. However, in all previously reported cases, this tail is of a considerably shorter length and does not interact with the N-terminus of a CS-related molecule. To the best of our knowledge, all known tails only interact with the closest monomer in the classical dimer orientation (A/B). An interaction between N- and C- termini is not possible due to the fact that dimeric ALDHs have a shortened N-terminus and tetrameric ALDHs do not contain a tail.C-terminal tails are not uncommon in ALDHs but are most often characterized in dimeric ALDH41 (PDB: 4QGK) and ALDH7A130 (PDB: 4ZUL). The FALDH contains a transmembrane helix (omitted from structure) and a gatekeeper helix which does not plug the active site entrance but merely caps it. ALDH7A1 is more similar in structure to ALDHTt as the \u201cCrook\u201d of the C-terminal tail plugs the substrate entrance tunnel firmly. Although dimeric ALDH possess a tail, it should be noted that in many cases the tail is not long enough to cap or plug the substrate access tunnel. In cases where it is long enough, it folds along the edge of the tunnel opening without ever interacting with the substrate entrance channel mouth residues.Two structures in the PDB show evidence for the tail of an ALDH being involved in active site access and regulation. These are membrane associated human FALDHTt could only be crystallized in a closed tail conformation in the apo and holoenzyme. The difference in resting conformations between ALDHTt and other ALDHs may be indicative of an allosteric regulation mechanism such as the model seen for non-phosphorylating GAPN16 or the requirement of stabilizing ligands between monomer interfaces42.In all tail containing ALDHs characterized thus far, the apo and cofactor bound proteins were in an open conformation. A closed conformation could only be found in product bound ALDH7A1. Surprisingly, ALDH43. We would like to note that our truncated mutants were of a much lower solubility than full length recombinant or native proteins highlighting the significance of the N- and C-termini interactions in the quaternary structure. We also investigated the tail extension as an obvious thermal clamp in a thermophilic genus such as Thermus however, no significant reduction in Tm (<5\u2009\u00b0C) was seen during thermal shift assays. This is unsurprising when it is noted that an ALDH of 515 residues in length exists in the close relative, T. thermophilus HB8. Such a protein would still contain a full complement of \u201cCrook & Hook\u201d tail residues allowing for active site regulation and may even perform more efficiently as it may be able to fold more easily into inter-domain clefts without the cumbersome longer tail.The longer tails in ALDHs were postulated to effect solubility in medically significant mutations such as c.1512delG which leads to pyridoxine-dependent epilepsyTt and its deletion mutant structures even without intermolecular interactions between the end of the tail and N-terminal interactions hints that active site access is regulated by additional domain interactions. It must be noted that the similar crystallization conditions and thus uniform space group may only allow for the closed and compact conformation of the protein. Phylogenetically, we hypothesize that the ancestral ALDH possessed both extended N- and C-termini, but either terminus was lost due to no evolutionary pressure being present in accordance with the potentially cumbersome regulation model needed in ALDHTt. Our structural data shows for the first time a clear rationale highlighting that ALDH first evolved with both extended N-termini and then diverged as either terminus was truncated.The similarity in tail orientation and active site regulatory loops between ALDHDeinococcus-Thermus phylum suggests that the presence of this C-terminal tail must have been chosen by evolution for specific functional reasons not, entirely, due to its habitat of high temperature. The very limited number of sequences with an extended c-terminal tail found outside the Deinococcus-Thermus phylum may argue for a phylum-specific adaptation that could have been subsequently lost over the course of evolution, especially considering that this phylum is rather ancient.The presence of longer tails in mesophilic bacteria belonging to the cf. for instance, ubiquinone:cytochromec oxidoreductase/bc1 complex44). Consequently, an alternative explanation must be considered \u2013 the extensive contacts made by the oligomerization domain potentially provide a structural stability even when the living conditions are not extreme. In this regard, the \u2018ancient\u2019 tetrameric assembly was not lost when this enzyme was retained in human and other mesophilic organisms.The oligomeric state of ALDH poses an obvious question as to the reason for the conservation of a tetrameric assembly even when the protomers are far apart in the three-dimensional so as to preclude any direct electron transfer  the tail swings away from the wall of the central pore attempting to allow substrate to enter into the monomeric subunit (2) the BOG cannot fit into the monomer so continues into the positively charged pore and (3) the tail closes again sequestering the detergent inside. One final possibility may be that the detergent has split the tetramer into dimers; however, this seems unlikely as there is no presence of detergent molecules on the other surfaces of the protein and BOG has been shown to be mild towards ALDH in the past. The presence of two conformations of detergent molecule most likely points to the two positions of the molecules when they enter from opposing interface tunnels. The negative head group of the detergent molecule is attracted to this charged pore and then becomes trapped in the central cavity. It is tempting to postulate that this highly charged pore is not coincidental and that this is a possible allosteric regulation site for ALDHTt which allows access to the substrate tunnel of each monomer.It could hardly be suggested that the BOG molecules entered the central pore of the tetramer Tt. The occlusion of the active site by the C-terminal arm in ALDHTt530 could be the reason for the increased activity in the truncated enzymes but the crook and hook fulcrums are still present in ALDHTt515 so this was expected to also have a similar activity to the recombinant wild type. The higher activity in the truncations may be explained by the easier folding of the tail away from the SEC like other ALDH discussed above but our structural data contradicts the movement. It may be the case that a folded or open crook and hook portion of the tail may exist during the in vitro assay, but the current crystallization conditions do not stabilize such a conformation during structural studies. Regardless, this higher activity is not observed at the higher temperature of 85\u2009\u00b0C as both mutants rapidly lost their function compared to ALDHTt530 where it was still active up to 4\u2009min, consistent with\u00a0the Tm of ALDHTt530 of 84\u2009\u00b0C.Interestingly, the truncated mutants were kinetically more active than\u00a0the full-length ALDHE. coli aldehyde dehydrogenase, AldB, has a KM of 5.8\u2009\u00b5M with the substrate propanal42, compared to the ALDHTt530 KM of 2.76\u2009mM (data not shown). Perhaps a more straightforward comparison would be with the enzyme from T. thermophilus HB8.Extrapolating our data herein to other previously-characterized enzymes is not possible as mesophiles and thermophiles have different optimal temperature. Another reason for a reserved comparison of ALDH is that the true substrate for many has not been identified and as such cannot be compared with ALDH kinetic data for enzymes with their actual physiological substrate defined. For instance, the extensively-studied T. thermophilus HB8, which forms a complex with an aldolase and occupies an interesting subclass of the ALDH superfamily50 also had its kinetic parameters defined at 25\u2009\u00b0C with a KM\u2009=\u20096.4\u2009mM compared to the ALDHTt530 KM\u2009=\u20092.76\u2009mM when propanal was used. However, a much more kinetically active enzyme exists in another thermophilic bacteria, Geobacillus thermodenitrificans, which is reported as having a KM of 6.6\u2009\u00b5M using acetaldehyde at 60\u2009\u00b0C14. Again, this increased kinetic ability may be due to the higher temperature but when attempted at 60\u2009\u00b0C we could not obtain reliable data due to the volatile nature of the aldehydes.An ALDH in T. thermophilus was identified as an impurity, via Edman degradation (deformylated) and mass spectrometry, during the crystallization of the caa3-cytochrome c oxidase whereby the oxidase and ALDHTt crystals grew in the presence of polyethylene glycol and ammonium sulfate as precipitating agents, respectively18. ALDHTt was purified from the caa3-oxidase by cation exchange chromatography and ammonium sulfate precipitation. Suitable bipyramidal crystals from 20\u2009mg/ml solution were obtained by the sitting-drop vapor diffusion method at 20\u2009\u00b0C, against 50\u2009mM MOPS pH 7.5 and 1.2\u2009M ammonium sulfate.Crystalline native ALDH from Tt  into the pET-22b (+) vector are summarized in Supplementary Table\u00a0Primers used for amplification and cloning of the gene encoding for ALDHTt were transformed into Escherichia coli BL21 Star (DE3) competent cells (Invitrogen). The auto-induction ZYM-5052 medium51, supplemented with ampicillin (100\u2009\u00b5g/ml), was inoculated with an overnight culture of the transformed cells at 1% (v/v), and grown for 48\u2009h at 25\u2009\u00b0C with shaking at 200\u2009rpm.The construct DNA for each of the wild type and truncated mutants of ALDHg, 15\u2009minutes, 4\u2009\u00b0C) and washed in 500\u2009ml of 20\u2009mM Tris-HCl pH 7.5, 5\u2009mM \u03b2-mercaptoethanol, 10\u2009mM imidazole and 500\u2009mM NaCl. Following a second centrifugation step, the cells were resuspended in the same buffer supplemented with Lysozyme (1\u2009mg/ml) and 5\u2009mM EDTA pH 8.0 and stirred at room temperature for 1\u2009h. DNase (0.1\u2009mg/ml) was then added along with 5\u2009mM MgCl2 and the cells were gently stirred at 4\u2009\u00b0C for 1\u2009h and sonicated in an ice-water bath for 20\u2009min. Finally, the mixture was heated to 65\u2009\u00b0C for 15\u2009min and centrifuged at 25,000\u2009\u00d7\u2009g, 30\u2009min, 4\u2009\u00b0C). The supernatant was then loaded onto an XK 16/20 column containing Ni Sepharose 6 Fast Flow  pre-equilibrated with 20\u2009mM Tris-HCl pH 7.5, 5\u2009mM \u03b2-mercaptoethanol, 10\u2009mM imidazole and 200\u2009mM NaCl. Bound proteins were eluted using a step gradient of 60, 100, 250 and 500\u2009mM of imidazole in 20\u2009mM Tris-HCl, 5\u2009mM \u03b2-mercaptoethanol and 150\u2009mM NaCl, pH 7.5. Fractions containing the protein were then dialyzed overnight in 50\u2009mM Tris-HCl, 5\u2009mM \u03b2-mercaptoethanol and 250\u2009mM NaCl, pH 7.5 prior to concentration using Amicon Ultra-15 centrifugal filters, 50\u2009kDa MWCO (Merck Millipore) and loaded onto a HiLoad 16/60 Superdex 200\u2009pg column pre-equilibrated with 50\u2009mM Tris-HCl pH 7.5, 5\u2009mM \u03b2-mercaptoethanol and 150\u2009mM NaCl.Cells were collected by centrifugation + and ALDHTt530 with propanal were achieved by adding 1\u2009mM of NAD(P)+ sodium salts and 50\u2009mM of propanal to the respective drops.All crystallization assays were realized in the same conditions as for native ALDH54 and high resolution cut off were selected according to Karplus & Diederichs55. For ALDHTtnative, 6 X-ray diffraction data were collected from multiple crystals, each having different effective maximum individual resolution as determined by the CC1/2 at the CORRECT stage. With the availability of multiple highly-diffracting isomorphous crystals, it was possible to merge and scale the datasets using XSCALE into a single dataset. Phasing was achieved through molecular replacement (phenix.MRage/Phaser)56 using multiple search models. The best solution was obtained with the human ALDH1A1 (PDB ID: 4WJ9)57 in the space group P41212. The partial model was automatically built using Phenix.AutoBuild58, improved manually with Coot59 and refined with phenix.refine60 and Refmac61. The obtained model was used to resolve the structure of the recombinant proteins. Phenix.Ligandfit was used to automatically place ligands in the structures. Data collection and refinement statistics are shown in Supplementary Table\u00a0Datasets were collected at the Diamond I04 and I24 beamlines. Data were processed using xia2/XDSTt samples. The dye Sypro orange (Invitrogen) and a qPCR machine (Roche Light cycler 480) with an excitation filter of 498\u2009nm and an emission filter of 610\u2009nm were used. Samples were equilibrated at 25\u2009\u00b0C for 3\u2009min before a ramped heating step between 25\u201395\u2009\u00b0C. The heating rate was set to 0.03\u2009\u00b0C/s corresponding to 20 acquisitions/\u00b0C. The first derivative and Tm were plotted using OriginPro 9.1.A thermofluor assay was used to assess the relative thermostability of the ALDH2PO4-K2HPO4 (Kpi) pH 8.0, 0.4\u2009mM NAD+, 60\u2009nM ALDH, 1\u2009mM hexanal, in a total volume of 0.5\u2009ml unless otherwise stated. A substrate-lacking reaction was used as a negative control. The reaction was monitored at 50\u2009\u00b0C for 60\u2009s. The reduction of NAD+ cofactor to NADH was monitored in a Cary fluorescence spectrophotometer  with respective slit widths of 20\u2009nm and 5\u2009nm. All components were heated before being added to the cuvette and the reaction was started with the addition of substrate. One unit of enzyme was defined as the amount of enzyme which catalyzed the formation of 1.0\u2009\u00b5mol of NADH/min. All assays were performed as a minimum of two independent experiments with triplicates for each reaction. Kinetics data and curve fitting were performed using OriginPro 9.1.Enzyme assays were performed over a short period of time to ensure no evaporative loss of the volatile aldehydes. The standard assay condition was chosen and optimized from the conditions which gave the largest and most stable change in the fluorescence readings. The standard enzyme assay consisted of 10\u2009mM KH62. The atomic coordinates and structure factors for ALDHTt native, ALDHTt530 in complex with propanoic acid, ALDHTt515 in complex with NADP and ALDHTt508 have been deposited in the Protein Data Bank www.pdb.org .All the figures were generated with Pymol  and CCP4mgSupplementary information"}
+{"text": "Herein, we put forward a tactic using soft crystalline cationic material with anion-adaptive dynamics for 99TcO4\u2212 sequestration. A cucurbit[8]uril-based supramolecular metal-organic material is produced through a multi-component assembly strategy and used as a sorbent for effective trapping of TcO4\u2212. Excellent separation of TcO4\u2212/ReO4\u2212 is demonstrated by fast removal kinetics, good sorption capacity and high distribution coefficient. Remarkably, the most superior selectivity among metal-organic materials reported so far, together with good hydrolytic stability, indicates potential for efficient TcO4\u2212 removal. The structure incorporating ReO4\u2212 reveals that the supramolecular framework undergoes adaptive reconstruction facilitating the effective accommodation of TcO4\u2212/ReO4\u2212. The results highlight opportunities for development of soft anion-adaptive sorbents for highly selective anion decontamination.Efficient anion recognition is of great significance for radioactive  4\u2212 ions.Efficient anion recognition and trapping is of great significance for anion-specific separation processes but the design of an anion-adaptive sorbents remains a challenge. Here the authors use a cucurbit[8]uril based soft cationic supramolecular material as efficient sorbent for TcO Complex chemical behavior of 99Tc hampers separation of uranium and plutonium during reprocessing of spent nuclear fuel, and high volatility of 99Tc species (Tc2O7) constrains incorporation into glass waste forms via high-temperature vitrification. 99Tc, as a stable TcO4\u2212 in its dominant\u2009+\u20097 oxidation state, is highly water soluble and can migrate readily in the environment, thereby posing severe environmental risks. Therefore, efficient capture of radioactive 99Tc has received considerable attention for both nuclear waste management and contaminant remediation purposes.4\u2212 from aqueous media6. In solvent extraction, extractants with anion recognition capability can achieve high selectivity for TcO4\u2212\u20099, but practical applications are limited by cost and inefficiency. Ion exchange is developed as an alternative of traditional extraction. Despite the ease of implementation and the expected efficient recovery of TcO4\u2212 based on ion-exchange method10, the total performance of sorbent materials used seems not to be competent. For example, most traditional polymeric materials exhibit slow anion exchange kinetics and poor radiation resistance12, while inorganic cationic materials such as layered double hydroxide (LDH)13, sulfides14, and borates16 exhibit low sorption capacity and poor selectivity. The emergence of hydrolytically stable cationic metal\u2013organic materials (MOMs) has led to potent applications for capture of oxyanion pollutants20. High porosity, structural diversity, and functional tunability23 render these hybrid materials as promising candidates for TcO4\u2212 removal25. However, there is still demand for improvement in terms of selectivity of TcO4\u2212 sorbents, with enhanced discrimination for TcO4\u2212/ReO4\u2212 over other anions as a particularly desirable attribute.Solvent extraction and ion exchange are two well-established effective methods for removal of TcO29 of TcO4\u2212 have the best ion selectivity. A rational approach for improving the selectivity of solid sorbents is to give sorbent materials an ideal ion-recognition capability by direct functionalization of traditional solid sorbents with covalently attached anion receptors . The CB8 macrocycle used here plays a vital role in accomplishing both the construction of a supramolecular network48 and anion recognition  flexibility of the CB8 encapsulation motif allowing dynamics of the overall supramolecular framework51.There is no doubt that anion receptorsors Fig.\u00a030,31, buelf Fig.\u00a032\u201336. Coion Fig.\u00a0, can be ion Fig.\u00a0, and end2@CB8)(H2O)4](NO3)2\u00b718H2O), is constructed by the supramolecular collaborative assembly. As expected, this material is demonstrated to be an efficient and selective sorbent capable of reversibly sequestrating TcO4\u2212/ReO4\u2212 by trapping them in specific tetrahedral pores created by CB8 moieties arranged in order. The anion-adaptive capability of this supramolecular sorbent toward effective TcO4\u2212 recognition resembles the dynamic behavior of the receptor during ion recognition, and can be taken as a representative TcO4\u2212-specific smart sorbent material.In this work, a CB8-based cationic supramolecular metal\u2013organic framework, SCP-IHEP-1 ([Cu((bpy)3)2 under hydrothermal conditions. It crystallizes in monoclinic space group P21/n  2@CB8]0.5\u00b719H2O 2. It is notable that, whereas 2G@H encapsulation, where G denotes a guest molecule, and H a host, is common for host CB852, this motif is rare for neutral guest such as bpy in CB853. Actually, formation of 2bpy@CB8 in aqueous solution involves a large favorable enthalpy change as a result of release of high-energy water  and Cu supramolecular framework via cross-linkage of a large number of interchain hydrogen bonds 2 in the absence of CB8 macrocycles results in a twofold interpenetrating 3D framework based on six-coordinated copper nodes with deformed octahedral geometry  remained essentially unchanged Fig.\u00a0. The res4\u2212 was initially used as a nonradioactive structural analog of 99TcO4\u2212 to assess anion exchange of SCP-IHEP-1. Batch kinetics experiment shows that removal of ReO4\u2212 by SCP-IHEP-1 follows the pseudo-first-order model -based MOF19, all of which take over 24\u2009h to reach exchange equilibrium for sequestering ReO4\u2212. It is notably that, SCP-IHEP-1 also shows faster removal rate and much higher removal ratio than its CB8-free counterpart Cu-bpy 13, NDTB-1 (49\u2009mg\u2009ReO4\u2212\u2009g\u22121)16, and UiO-66-NH3+ (159\u2009mg\u2009ReO4\u2212\u2009g\u22121)17. Assuming that all the nitrate ions can be exchanged, the sorption capacity of SCP-IHEP-1 for ReO4\u2212 observed here reaches to as high as 93% of the theoretical value (226\u2009mg\u2009g\u22121), suggesting its nearly perfect exchange tendency for ReO4\u2212. Moreover, the distribution coefficient (Kd) of SCP-IHEP-1 toward ReO4\u2212 is 2.6\u2009\u00d7\u2009105\u2009mL\u2009g\u22121  within a wide pH range of 4\u221210  during the sorption process with inorganic anion sorbents16. However, this order always is reversed for organic polymers and inorganic\u2013organic hybrid materials, which is taken as a Hofmeister phenomenon57. This Hofmeister behavior might be originated from the hydrophobic nature of organic backbones of these materials, as evidenced by the methylene/methylidyne-rich tetrahedral pores of SCP-IHEP-1.3 A similar trend is observed for other MOMs bearing local hydrophobic cavities or pores58. That is to say, considering the differences in hydration energy of anions, the preference for larger poorly hydrated ReO4\u2212 anions over NO3\u2212 or SO42\u2212 reflects the important role of hydration/dehydration in the anion exchange, which is consistent with the exothermic feature of exchange observed above.Removal selectivity of SCP-IHEP-1 toward TcO4\u2212 against NO3\u2212 and SO42\u2212 observed here is better than those of MOF-typed (SCU-101)25 and polymeric network-typed (SCU-CPN-1)57 anionic exchange materials with excellent ReO4\u2212/TcO4\u2212 removal performance emerging recently, making it a promising candidate for selective sequestration of TcO4\u2212 from waste solutions, even in the presence of high concentration of competing anions. To assess the potential application of SCP-IHEP-1 in real nuclear solutions containing radioactive TcO4\u2212, removal experiments for TcO4\u2212 were also tested. Uptake kinetics of TcO4\u2212 by SCP-IHEP-1 is as fast as that of ReO4\u2212, achieving ~80% removal at 1\u2009min and over 90% after 10\u2009min , although the TcO4\u2212 removal is affected by the high-concentration competing NO3\u2212, the removal percentage of TcO4\u2212 is still up to 79.2% using a solid-to-liquid ratio (SLR) of 0.5, which is superior to the removal by SCU-100 (59.3% with a SLR of 1.0)25 and SCU-101 (75.2% with a higher SLR of 10)24 and represents the best removal performance for 99TcO4\u2212 among cationic MOMs reported so far.The overall selectivity of SCP-IHEP-1 toward ReO4\u2212 (and by inference TcO4\u2212) exchange of SCP-IHEP-1 was monitored by Fourier transform infrared spectroscopy (FTIR)  were also obtained and subject to X-ray diffraction structural determination on the Beijing Synchrotron Radiation Facility (BSRF)  Fig.\u00a0, PXRD paIR) Fig.\u00a0, and eneIR) Fig.\u00a0. Single 4\u2212 is trapped in tetrahedral pores surrounded by four adjacent CB8 molecules   within SCP-IHEP-1 or SCP-IHEP-1-Re. The GGA-PBE59 functional implemented in VASP 5.460 was used to optimize the unit cells of the crystals, allowing relaxation of all the atom coordinates. With the fully optimized unit cells, three anion-containing tetrahedral pore models based on a simplified host system [H] consisting of the key components of the CB8 macrocycles were built  analysis was performed at the B3LYP-D3(BJ)/6-311+G level of theory (see Supplementary Methods for details). Several bond critical points (BCPs) between [H] and oxygen atoms of NO3\u2212 and ReO4\u2212 can be observed , Laplacian of electron density (\u22072\u03c1), kinetic energy density (G), and potential energy density (V) at the representative BCPs are listed in Supplementary Table\u00a0\u03c1\u2009<\u20090.07\u2009e/\u00c53, 0.2\u2009<\u2009\u22072\u03c1\u2009<\u20090.8\u2009e/\u00c55, 4.5\u2009<\u2009G\u2009<\u200918.3\u2009kJ/mol/Bohr3, \u221215.7\u2009<\u2009V\u2009<\u2009-3.1\u2009kJ/mol/Bohr3, respectively. For shorter H-Bond lengths , these values are within the scope of weak hydrogen bonding, while the larger ones belong to van der Waals interactions63. These intermolecular interactions can be further detected by independent gradient model (IGM)64 analysis and reduced density gradient (RDG)65 analysis  are similar for TcO4\u2212 and ReO4\u2212, and both of them are larger than that of NO3\u2212-[H] model (Supplementary Table\u00a0H] binding energies (\u0394BEgas) for all these three models are not significant, whilst the difference of hydration energy (\u0394Ehyd) between ReO4\u2212 and NO3\u2212 is dominant. This result suggests the vital role of hydrophobic nature of CB8-based methylene/methylidyne-rich tetrahedral pores of SCP-IHEP-1 in selective TcO4\u2212/ReO4\u2212 removal, and thus provides a valuable evidence for the Hofmeister bias selectivity of SCP-IHEP-1 mentioned before.We computed hydration energies, binding energies and total energies for anion exchange with uril (CB8) was synthesized according to the previously-reported literature3)2 aqueous solution  was added to a suspension of 4,4\u2032-bipyridine (bpy)  and CB8  in water (2\u2009mL) in a stainless-steel vessel. The mixture was sealed, and kept at 150\u2009\u00b0C for 48\u2009h. After cooling to room temperature, the obtained blue microcrystals of SCP-IHEP-1 were filtered, rinsed with water and ethanol three times, and dried in air at room temperature. Yield: 0.026\u2009g (59% based on CB8).An aliquot of 0.2\u2009M Cu(NO2@CB8]0.5\u00b7[(bpy)2@CB8]0.5\u00b719H2O : 8.49 ; 7.42 , 5.84 , 5.52 , 4.22 . (b) SCP-IHEP-1: 0.2\u2009M Cu(NO3)2 aqueous solution  was added to a suspension of 2bpy@CB8 intermediate obtained as described above in water (2\u2009mL) in a stainless-steel vessel. The mixture was sealed, and kept at 150\u2009\u00b0C for 48\u2009h. After cooling to room temperature, light-blue block crystals of SCP-IHEP-1 were obtained in a high yield .(a) 2bpy@CB8: 4,4\u2032-bipyridine (bpy)  was added to a suspension of CB8  in water (2\u2009mL). After incubation in a stainless-steel vessel at 150\u2009\u00b0C for 24\u2009h, colorless block crystals of 2bpy@CB8 were obtained in a quantitative yield during cooling to room temperature. The structure of 2bpy@CB8 was confirmed by single-crystal structure determination as [(bpy)O Figure\u00a0 and 1H-Na Figure\u00a0. 1H NMR 3)2 aqueous solution  was added to a suspension of 4,4\u2032-bipyridine (bpy)  in water (1.5\u2009mL) in a stainless-steel vessel. The mixture was sealed, and heated slowly to 150\u2009\u00b0C in a period of 24\u2009h, and kept at 150\u2009\u00b0C for another 48\u2009h. After slowly cooling to room temperature in a period of 24\u2009h, the obtained blue regular plate-like crystals of Cu-bpy were filtered, rinsed with water three times, and dried in air at room temperature. Yield: 0.013\u2009g.An aliquot of 0.2\u2009M Cu(NO4\u2212 (and by inference TcO4\u2212), crystals of ReO4\u2212-incorporated SCP-IHEP-1 sorbent were synthesized through an in situ assembly method. The detailed synthesis procedure is as follows: 0.2\u2009M Cu(NO3)2 aqueous solution  and 0.2\u2009M NH4ReO4 aqueous solution  was added to a suspension of 2bpy@CB8 obtained as described above in water (2\u2009mL) in a stainless-steel vessel. The mixture was sealed, and kept at 150\u2009\u00b0C for 48\u2009h. After cooling to room temperature, small light-blue prismatic crystals of SCP-IHEP-1-Re were obtained. The PXRD pattern of SCP-IHEP-1 after ReO4\u2212 uptake collected is fully consistent with the simulated PXRD pattern based on crystal data for SCP-IHEP-1-Re obtained above, suggesting identical structures.To elucidate the exchange and recognition mechanism for SCP-IHEP-1 with ReO3)2 aqueous solution  and 0.2\u2009M NH4ReO4 aqueous solution  was added to a suspension of 4,4\u2032-bipyridine (bpy)  in water (2.0\u2009mL) in a stainless-steel vessel. The mixture was sealed, and heated slowly to 150\u2009\u00b0C in a period of 24\u2009h, and kept at 150\u2009\u00b0C for another 48\u2009h. Blue block crystals of Cu-bpy-Re were obtained after slowly cooling to room temperature in a period of 24\u2009h.An aliquot of 0.2\u2009M Cu(NO\u03bb\u2009=\u20091.54178\u2009\u00c5) at room temperature. Data frames were collected using the program APEX 3 and processed using the program SAINT routine in APEX 3. Data collection for 2bpy@CB8 and SCP-IHEP-1-Re was acquired with synchrotron radiation at Beijing Synchrotron Radiation Facility  using a MAR CCD detector. The crystal was mounted in nylon loops and cooled in a cold nitrogen-gas stream at 100\u2009K. Data were indexed, integrated and scaled using DENZO and SCALEPACK from the HKL program suite. All crystal structures were solved by means of direct methods and refined with full-matrix least squares on SHELXL-9767, and refined with full-matrix least squares on SHELXL-201468. The crystal data of all compounds are given in Supplementary Table\u00a0X-ray diffraction data for SCP-IHEP-1, Cu-bpy, and Cu-bpy-Re were acquired on a Bruker D8 VENTURE X-ray CMOS diffractometer with a Cu K\u03b1 X-ray source  at a specified temperature , and separated with a 0.22\u2009\u03bcm nylon membrane filter. The concentrations of ReO4\u2212 in aqueous solution were determined by inductively coupled plasma optical emission spectrometry .All the sorption experiments were conducted using the batch sorption method. The solid/liquid ratio performed in all batch experiments was 0.5\u2009g\u2009LSupplementary Information"}
+{"text": "To evaluate face\u2010to\u2010face information provision in patient counselling for prenatal screening compared with two forms of digital information provision, namely, noninteractive instructional video or interactive video.We performed a prospective, noninferiority, cluster\u2010randomized controlled trial comparing face\u2010to\u2010face  with two forms of digital information provision (intervention) in counselling for prenatal screening. This study was performed in the Amsterdam UMC, the Netherlands, in 2017, and included women in the first trimester of pregnancy. Main outcomes were knowledge gained by the patient and counselling duration. We performed a noninferiority analysis.One hundred forty\u2010one women were included, randomized, and analysed. The baseline characteristics were comparable. The intervention group was noninferior compared with the control group regarding the level of satisfaction. The knowledge grade difference was higher after using intervention, and the duration was significantly longer in the face\u2010to\u2010face group at 23\u00a0minutes versus 16\u00a0minutes. The addition of interaction with the video made no difference in any of the outcomes.Adding an instructional video to patient counselling is of added value to improve patient's knowledge and shorten time consumption of the counsellor, therefore possibly saving costs. But this form of counselling maintains the same level of satisfaction. Counselling for prenatal screening is a complex process containing education, information, and evaluation in order to make a well\u2010considered decision.Counselling for prenatal screening has an increase in interdoctor variation and unpredictable time consumption.What is already known about this topic?Digital information provision added to face\u2010to\u2010face counselling shortens the counsellors' time significantly without decreasing satisfaction and even improving knowledge.Shortening the counsellors' time consumption can be a very cost\u2010effective way of saving time or increasing atient care.Adding interactivity to patient information provision does not improve knowledge or satisfaction.What does this study add? Counselling for prenatal screening is a complex process containing education, information, and evaluation in order to make a well\u2010considered decision.Counselling for prenatal screening has an increase in interdoctor variation and unpredictable time consumption.Digital information provision added to face\u2010to\u2010face counselling shortens the counsellors' time significantly without decreasing satisfaction and even improving knowledge.Shortening the counsellors' time consumption can be a very cost\u2010effective way of saving time or increasing patient care.Adding interactivity to patient information provision does not improve knowledge or satisfaction.1Prenatal screening was developed in the 1970s, as the result of medical innovations, such as invasive prenatal diagnostic tests and obstetrical ultrasound. Since then, prenatal screening has been a routinely offered medical test.A counselling consultation can be divided into two parts: information provision and the counselling itself. Since the counselling is complex and can have different requirements in different countries, such as face\u2010to\u2010face contact,There is yet another evolving area of innovation, which is the use of interactive electronic media to facilitate teaching and learning.In this study, we analysed whether an instructional video is noninferior to face\u2010to\u2010face information provision with regard to patient satisfaction. We also evaluated if video can be beneficial in regard to learning effect and duration of counselling. Moreover, we aimed to evaluate whether the addition of interactive elements to the instructional video is of added value.22.1We performed a prospective, noninferiority, cluster\u2010randomized controlled trial comparing face\u2010to\u2010face (control group) with digital (intervention group) information provision before counselling for prenatal screening. All eligible patients were invited in the outpatient clinic of the Amsterdam University Medical Centers, VUmc University Amsterdam, the Netherlands. Each consultation consisted of two parts: information provision and followed by face\u2010to\u2010face, personal counselling. The control group received usual care, meaning a single consultation of face\u2010to\u2010face information provision and counselling. The intervention group was randomized between information provision by means of an instructional or interactive video before they continued to face\u2010to\u2010face, personal counselling. Both groups had the same face\u2010to\u2010face component. The counsellors (n\u00a0=\u00a05) were experienced and trained according to the national guidelines (both midwives and doctors follow the same training and perform at least 50 counselling sessions per year), to inform the participants of the same content as that given in the video.In the trial, we included healthy women in the first trimester of their pregnancy, aged above 18\u00a0years, who had requested counselling for prenatal screening. The participants referred for an indication of increased risk of a chromosome abnormality in pregnancy were excluded. After filling out the informed consent form, the participants were given a short demographics questionnaire; they also completed a knowledge pretest, satisfaction questionnaire afterwards, and knowledge posttest directly after the counselling. The counsellors were blinded for the intervention randomization  outcome and received a satisfaction questionnaire after the counselling. Counsellors timed the duration of their total consultation. The participant watched or interacted with the video in a private waiting room immediately before the counselling. Women were asked to participate when they were 18\u00a0years or older, spoke the Dutch language, and came for routine prenatal screening counselling. They were excluded when they had an increased risk on chromosomal abnormalities. Thirty\u2010four women did not meet the inclusion criteria, mainly because of insufficient Dutch language skills. One hundred participants were lost in follow\u2010up at the 6\u2010week questionnaire, after which we decided not to use the related data for further interpretation. See Figure\u00a02.2Information provision and the counselling should usually be followed by a well\u2010informed decision on the part of the participant. All groups received both the same information and counselling in one consultation. The standardized information provision consisted of general information about the basic understanding of prenatal screening, the screening options, and the possible consequences of a negative or positive test result. The aim of the information provision was to educate the participant to enable her to make a proper, well\u2010informed decision during the counselling. This study addresses the knowledge participants gain and the satisfaction of the consultation, which are part of being well informed. After the counselling, we wished the participants to have insight into the following subjects: prevalence of trisomies in the general Dutch population, which chromosomal anomalies are tested for and how, which screening methods are offered, the difference between screening and diagnosing, the difference between invasive and noninvasive testing, and finally, the limitations of ultrasound screening. The video was based on a previously used group consultation presentation.http://eprint.elephantelearning.com/video\u2010page/Both the information provision and the counselling are needed to decide whether to undergo prenatal screening. Therefore, we carried out a pretest before the information provision and a posttest after the counselling. The key differences between the interactive and passive video were the mandatory questions that pause the interactive video. We added a progress bar, four pauses with written information, 10 multiple\u2010choice questions, and five stop/rewind popups elements to the video. Please find the instructional and interactive video here: 2.3Counselling took place 2\u00a0days per week. Because of logistics in the outpatient clinic, it was not possible to randomize all subjects every day, and we had random days of usual care and days of video information provision. On the video provision days, we computer randomized immediately before the counselling between an instructional (noninteractive) and an interactive video. The allocation was 1:1 for the control group and the intervention group and, again, 1:1 for the instructional video and the interactive video group. Participants were given an appointment at the desk for either of those days randomly (the administrative personnel were blinded). All counsellors were blinded for the randomization outcome.2.4Our primary outcome was the level of participant satisfaction after the entire consultation. Satisfaction was preferred over knowledge as primary outcome, because in case of counselling, it is more important to make a well\u2010informed, satisfying decision than to gain knowledge of the subject.2.5Data collection was conducted using research survey3Between August 2017 and December 2017, two hundred three women were approached and asked to participate. We stopped including participants when the sample size was reached. One hundred and sixty\u2010two women consented, and five were lost in follow\u2010up for the postcounselling questionnaire. These five participants  did not fill out their postcounselling questionnaire. We lost 10 participants in the control group and six in the intervention group because the counsellor did not fill out their questionnaire about these participants. We were able to analyse 67 women in the control group, 36 in the informative video group, and 38 in the interactive video group for the primary outcomes  and 3.93 , P\u00a0=\u00a0.00). After counselling, the knowledge grade increased in both groups. However, the difference between precounselling and postcounselling knowledge grade was significantly greater in the intervention group. The knowledge score before the counselling was not significantly different between the control and intervention groups. Counsellor satisfaction after the counselling was not significantly different.The duration of the counselling was 7\u00a0minutes shorter in the intervention . Thirdly, we were not able to have a proper 6\u2010week follow\u2010up and even had a loss in the counsellor questionnaire in follow\u2010up. We believe this is due to the fact that participants, and even counsellors, did not have enough commitment for this study to invest more time for the follow\u2010up. Finally, we did not properly pilot\u2010test to evaluate the video and the interactive version before using them. Therefore, improving the design of the intervention (by using the feedback from a pilot) could have improved the outcomes of the video and, especially, of the interactive video. This study can be interpreted as using a pilot instrument, of which we can work on further improvement.4.3Patient counselling is becoming increasingly important in shared decision making, and this study shows that digital media can make it more time effective and cost\u2010effective. The benefit of using an instructional video is that each participant will get the same information. Face\u2010to\u2010face information provision never guarantees this.The fact that adding interactivity to the video did not improve the experience is also in line with other studies showing inconsistent results from adding interactivity.5Our study shows that adding an instructional video to patient counselling is of added value to the time consumption of the counsellor, can save costs, and improves patient's knowledge but maintains the satisfaction of both patient and counsellor. Adding interactivity to the instructional video did not change these effects. There is still much to learn about patient counselling and education. Other methods should be evaluated, other educational strategies can be used, and further evaluation studies should always include an instructional design evaluation as well.None of the authors have any conflict of interest.R.D.L., P.C.B. and C.J.G. conceptualized the trial. Together with S.F.H., A.M.S. and F.S., they drafted the protocol, which was revised by all authors. R.D.L., S.F.H., and A.M.S. included all participants. R.D.L., S.F.H. and J.H. performed the statistical analysis. All authors read and approved the final manuscript.The study protocol was approved by the medical ethical committee of the VU University, Amsterdam, the Netherlands, with file ID 398 on August 2th 2017.Data are available on request from the authors."}
+{"text": "Beginning at the fifth hour after breakfast, H2 and CH4 concentrations significantly increased after wheat compared to rice and mung bean  (p < 0.05). Bloating and satiety scores significantly increased after wheat compared to rice (p < 0.05), and increased but did not reach statistical significance compared to mung bean (p > 0.05). A higher bloating score after wheat compared to rice and mung bean was observed clearly after lunch but not after breakfast. Conclusion: Wheat ingestion produced more intestinal gas and more bloating and satiety scores compared to rice and mung bean, especially after lunch. This provides insight into the role of intestinal gas in the development of bloating symptoms in IBS.The aim of this study is to evaluate the effect of rice, mung bean, and wheat noodle ingestion on intestinal gas production and postprandial gastrointestinal (GI) symptoms in non-constipation irritable bowel syndrome (IBS) patients. Methods: Twenty patients  underwent 8 h breath test studies and GI symptom evaluations after standard rice, wheat, or mung bean noodle meals at 8:00 a.m. in a randomized crossover study with a 1-week washout period. The same meal was ingested at 12:00 p.m. Results: The H Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder (FGID) that has chronic and disturbing effects on a patient\u2019s life. Complaints of gastrointestinal (GI) symptoms after food ingestion have been reported in 25\u201364% of IBS patients ,2,3. GasFoods can induce GI symptoms by several mechanisms, including exaggerated response to food, food allergy, food intolerances, and increased intestinal gas production . SymptomThe effects of food ingestion on clinical symptoms of IBS patients have mainly been studied in Western countries ,7,8. It Rice and wheat are major sources of carbohydrates. In healthy humans, rice is completely absorbed in the small bowel and produces less intestinal gas after ingestion compared to wheat and other sources of carbohydrates . A previ\u00ae, version 3.0.3, Monash University, Melbourne, Australia). The application indicates that mung bean contains a high amount of oligos-GOS and fructans and that intake should be avoided in IBS patients. However, oligosaccharides in mung bean are soluble in water and can be eliminated by adequate presoaking during the process of making the cellophane noodle [Cellophane noodle is a traditional Asian food. In Thailand and China, it is made from mung bean flour, a complex carbohydrate which is widely available across Asia. The Monash University FODMAP diet application classifies mung bean as a high FODMAP food item  patients, according to the Rome III criteria , were reThe patients with constipation-predominant IBS defined by Rome III criteria were identified and excluded from this study using a symptom questionnaire with the Bristol Stool Form Scale (BSFS).2) and methane (CH4) levels were blinded to the test meals. Exhaled breath specimens and GI symptom scores were acquired after the first standard meal at every 15 min for 8 h. The GI symptoms, including bloating, satiety, abdominal pain, abdominal burning, heartburn, urgency of stool, nausea, food regurgitation, acid regurgitation, belching, chest pain/discomfort, and flatulence, were evaluated using 10 cm visual analogue scales (VAS), where 0 indicated no symptoms and 10 represented the worst symptoms. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Faculty of Medicine, Chulalongkorn University (Project identification code 027/56).Participants were randomly assigned by block randomization to ingest three interventional meals  in a randomized crossover fashion with a one-week washout period. After overnight fasting, GI symptom scores and BSFS were assessed at baseline, then the study meal was given at 8:00 a.m. (breakfast) and 12:00 p.m. (lunch). Although the rice, wheat, and mung bean noodle looked different, all patients were unaware of the main component of the noodles, and the investigators who evaluated symptoms and measured the breath hydrogen .Every study meal was made from 90 g dry weight of rice, wheat, or mung bean flour and cooked in noodle form. No vegetables or other poorly absorbed ingredients were added so as to avoid intestinal gas production from other sources. For each study, the same noodle meal made from rice, wheat, or mung bean was given for breakfast and lunch in a randomized cross-over fashion. A glass of water (240 mL) was ingested with the test meals. No food or drink was allowed between meals or after lunch. According to the United States Department of Agriculture (USDA) Food Composition Databases, the total energy in each serving size (90 gm dry weight) for rice, mung bean, and wheat noodle was 327.6, 319.5, and 324.9 kcal, respectively. The amounts of CHO:protein:fat content in a serving size of rice, mung bean, and wheat noodle were 72.2:5.4:0.5 gm, 77.5:0.14:0.05 gm, and 65.3:10.8:1.49 gm, respectively . The amount of H2 and CH4 in a breath sample were reported in parts per million (ppm).Breath samples were collected using a 250 mL sample holding bag . Subjects were asked to maintain good oral hygiene during the breath testing phase by brushing their teeth before taking their first breath sample and to refrain from vigorous physical activity . On the 2 and CH4 production after rice, wheat, and mung bean noodle ingestion evaluated from the breath samples collected over an 8 h study period after breakfast. Secondary endpoints were GI symptom score differences after the ingestion of different interventional meals. The sample size was calculated based on the best data available from a previous study of H2 production after rice ingestion in healthy volunteers [The primary endpoint was the difference between the intestinal Hlunteers . To dete2, CH4, and GI symptoms involving more than 2 groups were analyzed by the repeat-measures ANOVA and Wilcoxon signed-rank test for parametric and non-parametric data, respectively. A p-value of less than 0.05 was defined as statistically significant. Data were expressed as mean \u00b1 SD, unless stated otherwise. The data were analyzed using SPSS software version 17.0 for Microsoft Windows.Comparison of H2. All patients completed the studies without any adverse events. All subjects completed the assigned study meals. The patients\u2019 baseline symptom scores are shown in p > 0.05).Twenty non-C IBS patients  were included. The duration of IBS symptoms before the first diagnosis of IBS was 10.6 \u00b1 0.9 months, and the patients\u2019 BMI was 23.3 \u00b1 4.0 kg/m2 and CH4 concentrations in breath samples was similar at baseline before breakfast ingestion when comparing the rice, wheat, and mung bean (p > 0.05) (2 and CH4 concentration in breath samples for rice noodles were significantly lower than those of wheat noodles (p < 0.05), and the difference of H2 and CH4 concentration persisted until 450 min and 420 min, respectively, after breakfast ingestion. However, H2 and CH4 concentration in the breath samples for mung bean noodle were significantly lower than those of wheat noodle beginning from 240 min and 270 min until 450 min and 465 min after breakfast ingestion, respectively (p < 0.05). The H2 and CH4 concentrations in breath samples after mung bean and rice ingestions were similar throughout the study period (p > 0.05) (The H > 0.05) . Beginni > 0.05) .2 and CH4 concentrations over 8 h after wheat noodle ingestion were significantly greater compared to those after rice and mung bean noodle ingestion (p < 0.05) (2 levels after wheat ingestion were significantly higher than those after rice and mung bean ingestion (p < 0.05). Likewise, the maximum CH4 levels after wheat ingestion were significantly higher than those after rice ingestion, but did not reach statistical significance compared to mung bean (The mean and area under the curve (AUC) of H < 0.05) . The maxung bean .p < 0.05). Although the bloating and satiety symptom scores were higher after wheat ingestion compared to mung bean, the difference did not reach statistical significance   .p > 0.05) (p < 0.05) after breakfast.When the bloating symptom severity scores were plotted over time after breakfast ingestion, the bloating symptom scores increased immediately after each meal compared to before the meal, with a greater increase of symptom scores after lunch compared to breakfast, but did not reach statistical significance ( > 0.05) . The blop > 0.05) and gradually decreased toward the next meal. The improvement of satiety symptom severity scores were similar after breakfast, but after lunch, the improvement was significantly slower for wheat in relation to rice, leading to significantly higher satiety symptom scores after wheat ingestion compared to rice at 330, 345, 360, 375, 390, 420, and 435 min after breakfast, respectively (p < 0.05).Other GI symptoms, including abdominal pain, abdominal burning, nausea, urgency of defecation, heartburn, belching, and regurgitation, were not significantly different between rice, mung bean, and wheat after ingestion .2 and CH4 production of every 15 min time period (defined as the AUC of each 15 min period) evaluated during the 8 h study periods correlated significantly with the corresponding bloating symptom severity score after the wheat test meal , but there was no significant correlation with rice  or with mung bean . There was no significant correlation between H2 and CH4 production and satiety symptoms after wheat, rice, and mung bean meals .The H2 and CH4) production and GI symptoms for 8 h after breakfast and lunch. We found that rice and mung bean noodles produced similar levels of H2 and CH4 in breath samples and a similar severity of postprandial gastrointestinal symptoms, whereas wheat noodles produced higher levels of intestinal gas and a higher bloating symptom severity score compared to rice and mung bean. The satiety symptom scores also significantly increased after wheat noodle ingestion compared to rice noodle ingestion, and increased but did not reach statistical significance compared to mung bean noodle ingestion.Our study investigated the effects of the ingestion of 3 common carbohydrate sources in noodle form\u2014rice, mung bean (cellophane), and wheat\u2014on intestinal gas production and postprandial gastrointestinal symptoms in non-C IBS patients. We performed simultaneous monitoring of intestinal gas  from the ileum into the large bowel stimulated by lunch ingestion. The bloating symptom score increased immediately after breakfast to a similar degree between rice, mung bean, and wheat, and gradually decreased towards the next meal (lunch)  . ImmediaThe finding that, after lunch, the improvement of satiety symptom after wheat was delayed compared to rice suggests that an increase in intestinal gas may prolong postprandial satiety symptoms.2 and CH4, than rice and mung bean noodles. The low intestinal gas production after mung bean noodle ingestion found in this study indicates the elimination of oligosaccharides by adequate presoaking during the process of making mung bean noodle, whereas the wheat noodle preparation process did not provide adequate elimination of the FODMAP contents.The Monash University FODMAP diet application classified rice as a low FODMAP food. In contrast, mung bean and wheat are classified as high FODMAP foods. Rice and mung bean are major sources of carbohydrates in Asia, whereas wheat is a major source of carbohydrates in Western countries . We founThe different effects on intestinal gas production and gastrointestinal symptoms of rice and mung bean compared to wheat could be seen at the beginning of the fifth hour after breakfast ingestion. This result agrees with other studies that demonstrated the positive effects of a low FODMAP diet on IBS symptoms that appear early, within 1 or 2 days .Rice, wheat, and mung bean are 3 major sources of carbohydrates. Rice and wheat rank high on the glycemic index ,24, wherPrevious studies showed that GI symptoms, especially bloating and early satiety, after wheat ingestion resulted not only from incomplete intestinal absorption but also from osmotic load and luminal distention in the small or large intestine, which can trigger symptoms by increasing small bowel water content, confirmed by abdominal MRI .The limitations of our study are: (1) we could not make the 3 different study meals look similar; (2) ingestion of meal in the evening before the study date may affect intestinal gas production after breakfast in this study; (3) the sample size of 20 was relatively small to demonstrate the difference in severity of some symptoms; and (4) the amount of CHO:protein:fat per gram dry weight in rice, mung bean, and wheat noodle was not identical. However, we tried to minimize these limitations by not informing the patients of the CHO sources and expected effects, using a crossover method with a 1-week washout period, by blinding the study meals to the investigators, and by asking the patients to record their diet on the day before the study. The fact that there is less CHO is in wheat noodle than rice and mung bean noodle, which supports our conclusion that wheat CHO produces more gas than rice and mung bean, is not a limitation.We excluded patients with constipation because it has been reported that constipation patients may harbor preformed gas in hard stools, and the gas can be released when mixing of the intestinal content is induced . In our In conclusion, this study suggests that rice and mung bean are better sources of carbohydrates for non-C IBS than wheat carbohydrate, and they produce less intestinal gas and fewer bloating and satiety symptoms. Increased intestinal gas production after wheat, a high FODMAP food item, developed obviously after the second meal (lunch ingestion) and was associated with more postprandial bloating and satiety symptoms after lunch. This study provides insight into the role of intestinal gas production on the development of postprandial bloating and satiety symptoms in non-C IBS patients."}
+{"text": "Cycadopites and Praenymphaeapollenites cenomaniensis gen. and sp. nov., a form-taxon of pollen from a basal angiosperm lineage of water lilies . We demonstrate how a gymnosperm to angiosperm plant-host shift occurred during the mid-Cretaceous, from a generalist pollen-feeding family of beetles, which served as a driving mechanism for the subsequent success of flowering plants.The Cretaceous fossil record of amber provides a variety of evidence that is essential for greater understanding of early pollination strategies. Here, we describe four pieces of ca. 99-million-year-old (early Cenomanian) Myanmar amber from Kachin containing four closely related genera of short-winged flower beetles (Coleoptera: Kateretidae) associated with abundant pollen grains identified as three distinct palynomorphotypes of the gymnosperm   \u2022A gymnosperm to angiosperm plant-host shift is denoted during the mid-Cretaceous\u2022Kateretidae beetles are among the earliest pollinators of angiosperms\u2022Mesozoic direct evidences of angiosperm pollination just started to arise\u2022Praenymphaeapollenites is defined as a new angiosperm pollen morphotype Biological Sciences; Evolutionary Biology; Evolutionary Ecology; Paleobiology For much of land plant history, terrestrial vegetation consisted of free-sporing plants such as mosses, lycopods, ferns, horsetails, and gymnospermous seed plants whose mid-Mesozoic representatives included overwhelmingly conifers, cycads, ginkgoaleans, czekanowskialeans, corystosperms, caytonialeans, bennettitaleans, and gnetaleans . AngiospMutualisms between insects and plants are among the most thoroughly studied of organismic interactions . The oriAlthough wind pollination is the prevalent mode of pollination in extant gymnosperms, field observation and experimental studies have documented insect visitation in a number of cycad species and in all genera of Gnetales . EndressIn this report, we provide the first direct evidence that closely related species of short-winged flower beetles (Coleoptera: Kateretidae) fed on and transferred pollen from three different gymnosperm plant hosts and an early angiosperm plant host at the Early Cretaceous-Late Cretaceous boundary interval (99 mya). This breadth of pollinated seed plants by a lineage of closely related beetle taxa constituted an initial step in the transition from a gymnosperm-beetle to an angiosperm-beetle mutualism by a simple shift of a host plant from a gymnosperm to an angiosperm. The gymnosperm hosts (as pollen) were cycads, ginkgoaleans, and bennettitaleans, whereas the angiosperm host  was a water lily . Nymphaeales share a sister group or an adjacent paraphyletic relationship with Amborellaceae, the basalmost extant lineage of angiosperms. The closeness of these mutualisms provides a major advance for understanding the mode in which the transition from gymnosperm hosts to a basal angiosperm occurred in one particular insect pollinator family of beetles within the same local environment. We note that, to our knowledge, this is also the first evidence of a fossil association likely engaged in aggregative behavior.Electrumeretes birmanicus, Polliniretes penalveri, Cretaretes minimus, and Eoceniretes antiquus  that we examined, in contrast to the other pollen samples containing rphotype F\u20132I. Thirphotype .Electrumeretes\u00a0birmanicus occurs isolated as one beetle in NIGP171364 together with an indeterminate beetle specimen as syninclusion  polGymnospermae Cycadopites sp1 (MGB 87960) (B 87960) ADescription: Pollen grains subprolate to rounded; 20.89\u00a0\u03bcm in length \u00d7 18.42\u00a0\u03bcm in width on average; range in length: 18.57\u201322.85\u00a0\u03bcm; range in width: 14.28\u201322.85\u00a0\u03bcm; monosulcate; sulcus elongate for the entire length of the grain; sulcus margin folded; exine; 1\u20132.5\u00a0\u03bcm thick; surface psilate.Cycadopites sp2 (MGB 87961) (B 87961) BDescription: Pollen grains prolate; 18.41\u00a0\u03bcm in length \u00d7 10.41\u00a0\u03bcm in width on average; range in length: 15.71\u201321.42\u00a0\u03bcm; range in width: 7.14\u201312.85\u00a0\u03bcm; monosulcate; sulcus elongate for the entire length of the grain, sometimes wider at their ends and constricted toward their equatorial area; sulcus margin folded; exine around 1.5\u00a0\u03bcm thick; surface psilate.Cycadopites sp3 (NIGP171364) Description: Pollen grains prolate, sometimes subprolate; 26.27\u00a0\u03bcm in length \u00d7 14.84\u00a0\u03bcm in width on average; range in length: 21.42\u201332.14\u00a0\u03bcm; range in width: 10.71\u201317.85\u00a0\u03bcm; monosulcate; sulcus elongate for the entire length of the grain; sulcus outline oval, sometimes wider at their ends and constricted toward their equatorial area; sulcus margin folded; exine 1.5\u20132\u00a0\u03bcm thick; surface psilate. Pollen grains apparently are isolated or integrated in clusters of more than 15 specimens  from late Albian amber of northern Spain  (a NIGP17165 (3)Type species: Praenymphaeapollenites cenomaniensis gen. and sp. nov. Barr\u00f3n, Peris and Labandeira.Etymology: The generic name is derived from the prefix \u201cPrae\u2013\u201d indicating a previous find, and the root \u201c\u2013nymphaeapollenites,\u201d the most similar fossil pollen type, known only from the Cenozoic.Diagnosis: Pollen grains zonasulculate, outline of the equator circular to subcircular, exine tectate, and strongly columellate; surface covered by evenly spaced thick verrucae as well as scarce short baculae sometimes ordered as an irregular reticulum-like pattern.Description: Pollen grains zonasulculate, usually grains show broken sulculus being divided in two halves; operculum is lacking or cannot be observed; grains isolate or aggregate in clusters; outline of the equator circular to subcircular; longer equatorial axis 60.67\u00a0\u03bcm on average (range 42.85\u201385.71\u00a0\u03bcm); exine tectate and strongly columellate; endexine thin, 1\u20132\u00a0\u03bcm thick; ectexine columellate around 3\u20135\u00a0\u03bcm thick; columellae >1\u00a0\u03bcm width; thick verrucae usually appears in the surface at the top of each columellae; verrucae and in lesser extent baculae ornamentation; surface covered by spaced thick verrucae as well as scarce short bacula sometimes ordered in irregular reticulum-like pattern; verrucae 1\u20132\u00a0\u03bcm in basal width.Praenymphaeapollenites cenomaniensis sp. nov. Barr\u00f3n, Peris and Labandeira , Hukawng Valley, Noije Bum Range, from Kachin State, northern Myanmar; Late Cretaceous (Early Cenomanian) in age  in age .Diagnosis: As for the genus, additional characteristics include the following: longer equatorial axis 60.67\u00a0\u03bcm on average (range 42.85\u201385.71\u00a0\u03bcm); endexine thin, 1\u20132\u00a0\u03bcm thick; ectexine columellate around 3\u20135\u00a0\u03bcm thick; columellae >1\u00a0\u03bcm width; verrucae 1\u20132\u00a0\u03bcm in basal width.Description: As for the genus, measurements of the pollen are in Remarks: The pollen examined in NIGP171365 resembles grains of extant species of Nymphaea such as Nymphaea alba and Nymphaea odorata. This pollen also shows similarities in shape and aperture structure to the Neogene species described as Nymphaeacidites by Nymphaeapollenites. Although Nymphaeapollenites, by considering only pollen grains with psilate surfaces from zonasulcate specimens of the German Miocene, Nymphaeapollenites is well differentiated from Nymphaeacidites by the lack of reticulate ornamentation. Cenozoic and recent grains are clearly distinguished from the examined Cretaceous ones by their finely columellate exines. Nuphar and Nymphaeoideae, which is \u201cintermediate\u201d between the granular and columellar condition, was derived from a columellar pollen. The presence of a tectate-columellate exine with supratectal verrucate and baculate surface ornamentation in the examined specimens allows us to infer that P. cenomaniensis sp. nov. was a precursor taxon to extant Nymphaeaceae.The oldest fossil record of nymphaealean plants is supported by Early Cretaceous leaves, flowers, and fruits . For polOccasionally, the fossil record reveals evidence of pollinivory, or pollen consumption, which consists of pollen present in insect guts, or more remotely, pollen found in coprolites in which the identities of the insect consumer and consumed pollen often are known. However, pollinivory is not coextensive with pollination, as some palynivores are not pollinators, and vice versa. Nevertheless, pollen consumption often has been the evolutionary precursor to biotic pollination . AlternaPollination relationships among diverse insect and gymnosperm groups since the Late Paleozoic have been suggested by substantial but indirect paleontological evidence . IndirecAfropollis   . In 105-ean host . A long-titalean . An Oeden grains . In addin grains , and a ln grains . Angiospn grains , althougroidites  and a moroidites , both alroidites , descriproidites . By controidites .Figure\u00a04Kateretidae (short-winged flower beetles) are a family of beetles (Coleoptera), members of the suborder Polyphaga, which range from small to medium in size and at present consist of 14 genera and approximately 95 species occurring in subtropical to temperate zones of the northern and southern hemispheres . Larvae Amborella-like lineage, probably during the Barremian to early Aptian interval (The Nymphaeaceae (water lily) together with the Cabombaceae (water shields) and the Hydatellaceae, constitutes the Nymphaeales, a clade of aquatic plants that is a sister group or adjacent paraphyletic group to extant Amborellaceae , the phyinterval . A younginterval  has beeninterval . Likewisinterval , Albian interval , Turoniainterval , and theinterval . Howeverinterval . Althouginterval .Nympheaceae are currently herbaceous insect-pollinated angiosperms defined by a distinctive morphology. They possess rhizomatous roots; stems that bear vascular bundles, prominent air-filled canals, often lactifers, and simple, mucilage-producing hairs; and leaves that are typically whorled on the stem and have long petioles that are submerged, floating, or emergent with palmate or pinnate venation . The floExtant water lilies occur worldwide in temperate to tropical climates in standing freshwater ecosystems . Such anGinkgo (Of the four major lineages of current gymnosperms, wind pollination is the exclusive pollination mode in extant conifers and Ginkgo , whereasGinkgo . For theGinkgo . Most seGinkgo . It has Ginkgo . EntomopGinkgo . Crepet Ginkgo . Long-prGinkgo . NotablyGinkgo . The extGinkgo . In distGinkgo . The evoGinkgo . By CenoGinkgo .Early angiosperms evolved in an environment where small insects such as moths, thrips, beetles, flies, and wasps were feeding on a variety of seed-plant lineages as the sole hosts, including ginkgoaleans, bennettitaleans, and cycads . The relThe study of pollen grains requires observation under high-magnification lenses; however, most pollen grains are badly preserved and important characters of the grains are hardly found. The observation of different grains most exposed at the amber surface was required to completely describe the pollen characters. Furthermore, several grains are in contact with the body surface of the specimens, although in areas with limited access to be illustrated.All methods can be found in the accompanying"}
+{"text": "Ganglion cysts (GCs) are tumor-like lesions that often occur in the soft tissues, which are mostly caused by the degeneration of mucin produced by the joint capsule and tendon sheath on the carpal dorsal joints of extremities. GCs may appear asymptomatic as benign tumors, but some patients also seek treatment because of the pain caused by these fluid-filled cysts. As a kind of complementary and alternative therapy, there have been some studies published in China which have proved that the fire needle has a better therapeutic effect on ganglion cyst. The purpose of this systematic review is to evaluate the efficacy of fire needle in the treatment of GCs.PubMed, EMBASE, the Cochrane Library, Chinese National Knowledge Infrastructure, Chinese VIP Information, Wanfang Database, and Chinese Biomedical Literature Database were searched by 2 reviewers from the inception until August 2020. The original study that randomised control trials of fire needle for GCs will be selected and is not limited by country or language. In addition, researches in progress, the reference lists and the citation lists of identified publications will be retrieved similarly. Study selection, data extraction, and assessment of the quality will be performed independently by 2 reviewers who have been trained prior to data extraction. A meta-analysis will be conduct if the quantity and quality of the original studies included are satisfactory; otherwise, a descriptive analysis will be conducted. Review Manager V5.4:  software will be using for data synthesis and assessment the risk of bias according by Cochrane Handbook.This study will provide a comprehensive review of current evidence for the treatment of fire needle on GCs.The conclusion of this study will provide a judging basis that whether the treatment of GCs with fire needle is effective.INPLASY202080032 Most GCs occur in the wrist. Dorsal wrist GCs account for 60% to 70% of all GCs, with volar wrist GCs accounting for about 18% to 20%. GCs typically consist of a cyst sac and the cyst may have a single cavity or multiloculated. Under B-ultrasound, the cyst wall is thin and smooth, with clear boundary, showing medium or high echo, and the interior shows low echo shadow. GCs are not considered true cysts because they lack a cellular epithelial lining, seen in synovial tissue or adventitial bursa.Ganglion cysts (GCs) is a cystic tumor-like lesion with pain as the most common symptom. Spherical masses can be observed at the body's surface, which is delimited by dense connective tissue and filled with gelatinous fluid that made up of glucosamine, albumen, globulin, and a high concentration of hyaluronic acid and the patient's pain of hand or wrist is mainly caused by these fluid-filled cysts. So there is no definitive treatment for the pathogeny. In order to relieve the patient's pain, many treatments have been adopted, non-operative treatment includes supportive splinting, non-steroidal anti-inflammatory drugs and aspiration,,6 and surgical procedures, etc. These therapies are successfully employed in clinical practice for GCs treatment, but the treatment effects are not always Satisfactory with the long treatment cycle and high recurrence rate. Recurrence of the cyst following surgery has been reported to range from 4% to 40%. Therefore, in recent years, more and more studies tend to evaluate the efficacy and safety of complementary and alternative therapies in the treatment of GCs.The pathogenesis of GCs is not clear, but it is usually not caused by a single factor. The cause of its occurrence has included congenital anomaly already, also be the stimulation that receives local stress possibly.,9 As an non-drug therapy, fire needle has been reported in some clinical studies that has certain curative effect on GCs. Therefore, the purpose of this study was to summarize the original research on the treatment of GCs with fire needle, so as to evaluate whether the treatment of GCs with fire needle is really effective.In China, acupuncture and moxibustion are effective traditional therapeutics and fire needle is an operation method in traditional acupuncture therapy. By stimulating the acupuncture points, the fire needle can dredge the meridians, accelerate the flow of qi and blood, and make the cyst dissipate.22.1 It is registered in the INPLASY .This protocol will be reported according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses Protocols.2.2PICOS will be applied, including Population, Intervention, Comparison, Outcome, and Study.2.2.1Randomized controlled trials (RCTs) with fire needle as the primary intervention for GCs will be included, and other studies such as case reports, and reviews will be excluded. No restrictions on country but language will be limited on English and Chinese.2.2.2 will be included. No restrictions on gender, age, race, etc.Participants diagnosed as GCs by clinicians referring to the New Routine for Diagnosis and Treatment2.2.3Without limits on course and dose, we will include studies in which fire needle is the primary intervention and, if necessary, we will include studies in which fire needle is combined with other active treatments versus active treatment alone.2.2.4The selected RCTs should testify that the interventions were compared with a control group composed of placebo, sham acupuncture, no treatment, or other active therapies.2.2.5Primary outcome: effective rate and the cyst diameter.Secondary outcomes: recurrence rate; adverse events incidence caused by fire needle, such as dizziness, vomiting, weariness, etc.2.32.3.1Two investigators will retrieve the relevant RCTs in the following databases: PubMed, Embase, the Cochrane Library, CNKI, Chinese VIP information, Wanfang Database, and CBM, from inception until August 2020 without restriction to publication status and languages. A comprehensive search strategy will be conducted, various combinations of MeSH items and free words will be searched synchronously, including \u201cganglion cysts\u201d, \u201cTendon sheath cyst\u201d, \u201cfire needle\u201d, \u201chuo zhen\u201d and etc. The preliminary search strategy for PubMed is presented in Table 2.3.2The relevant published references and citation list will be retrieved in Web of Science. In addition, the relevant systematic reviews or overview will also be identified for additional relevant studies. Moreover, relevant paper versions of medical journals and journals will be screened to ensure that the original studies that not included in the electronic databases could be included possibly.2.42.4.1All reviewers undergo rigorous training prior to selecting the study. Preliminary screening of the study will be conducted by 2 reviewers independently. After searching, the duplicated studies will be removal initially from the retrieved studies by Endnote (X9). And then, 2 independent reviewers (JC and LBL) will screened titles, abstracts, and keywords of all retrieved studies for candidates according to the inclusion and exclusion criteria, we will obtain the full text of all possibly relevant studies. Excluded studies will be recorded with explanations. If it is uncertain whether to adopt because of the lack of information, LBL will try to contact authors of the original reports to obtain the information of lost. During the procedure, disagreements will be resolved by discussion or consensus with the third reviewer (JX). Study selection will be performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses flowchart Fig. .2.4.2A unified data extraction table will be designed before data extraction, and data extraction will also be carried out independently by 2 reviewers (SYZ and GHT). The proposed extracted information includes:(1)General information: author, country, year of publication, study design, and database;(2)Population characteristics: sex, age, baseline diseases, and sample size;(3)methodological characteristics: information sources, intervention(s), comparison(s), bias assessment, etc. Any objections will be discussed by 2 reviewers, and further objections will be arbitrated by the third author (ZYZ).2.4.3 The specific evaluation items include the following 7 aspects: generation of random sequence, allocation concealment, blindness of participants, and personnel, blindness of outcome assessment, incomplete outcome data, selective reporting and other bias.To systematically evaluate the quality of each of the studies that final included. Two reviewers (SYZ and LBL) will assess the risk of bias for each included study according to the Cochrane handbook. It will eventually be rated on 3 levels .2.4.4Review Manager (RevMan\u200aV 5.4) will be used for data analysis and quantitative data synthesis. We will use the weight mean difference and 95% confidence interval to measure the continuous variables, while the results of dichotomous variables will using risk ratio and its 95% confidence interval.2.4.5 For the data missing at random, the analysis will rely on existing data, while we will filling the missing data with replacement values and make a sensitivity analysis to examine the potential impact of missing information, if necessary.If the specific information we need to collect are not be reported, the reviewer (GHT) will attempt to contact the original author for relevant information by telephone or e-mail. If the required information is not available, it will be explained in the article. Then, the missing data will be assumed to be \u201cmissing at random\u201d and \u201cmissing not at random\u201d according to the Cochrane Handbook.2.4.6 and the value of I2 represents the heterogeneity after data synthesis. We will use I2 to assess statistical heterogeneity between trials. If the I2\u200a<\u200a50%, that indicates slight or no heterogeneity in the evidence of the combined results, while I2 \u2265 50%, it means studies with high heterogeneity. The fixed effects model will be adopted when the P\u200a>\u200a.1 and I2\u200a<\u200a50%, while apply the random effect if P\u200a<\u200a.1 and I2\u200a\u2265\u200a50%.Heterogeneity refers to the difference between studies in the systematic review,2.4.7An assessment of the reported bias will be presented in the form of a funnel plot. If the points on both sides of the funnel plot are scattered and asymmetric, it is considered that there is a report bias and the reliability of this study is low. On the contrary, if the point distribution on both sides of the funnel plot is symmetrical, we believe that there is no or very low reporting bias, and the results of this study are reliable.2.4.82 obtained after data consolidation is greater than 50% and the P value is less than .1, sensitivity or subgroup analysis will be performed to exclude the source of heterogeneity. If the included original research data is insufficient for quantitative analysis, the review will only represent, and summarize the evidence.All analysis will be done through RevMan 5.4. According to heterogeneity assessment, mean difference or relative risk were calculated using fixed or random effects models. In addition, if the I2.4.9If the results show significant heterogeneity and the number of included studies is sufficient, sensitivity analysis will be performed to identify the quality and robustness of the meta-analysis result, which includes assessing the impact of sample size, methodological elements and the characteristic of research, and missing data.2.4.10 The quality of evidences will be rated on 4 levels . Two reviewers (XCZ and HG) will conduct the assessment process separately and describe in detail the reasons for downgraded or upgraded outcomes affecting the quality of evidence to guarantee the reliability and transparency of results.The quality of evidence will be evaluated using the Grading of Recommendations Assessment, Development, and Evaluation.3 they often cause pain, leading to significant motor dysfunction in the affected joint, which in turn reduces the patient's quality of life. Because the pathogenesis is not clear, according to the existing treatment principles, drug treatment is mainly non-steroidal anti-inflammatory drugs injection, non-drug treatment is mainly puncture aspiration, and surgical treatment. However, drug therapy is often accompanied by certain side effects, and the postoperative recurrence rate is high. As a result, many patients are looking for easier and less harmful alternatives.GCs are the most common tumors of the hand. Although they rarely deteriorate, In recent years, a certain amount of studies conducted in China have shown that compared with conventional puncture aspiration and steroid administration, fire needle has a higher cure rate for the treatment of GCs.As an alternative therapy for external therapy, the fire needle has a history of nearly 3 thousand years in China. It can relieve pain, improve the blood circulation, stimulate metabolism of local tissue.However, the efficacy of fire needles in treating GCs has been controversial due to the lack of evidence-based medicine, and some studies have reported that acupuncture may be a placebo effect. To date, there is no reliable comprehensive review of the treatment of GCs with fire needle. We conducted this study to assess the efficacy of fire needles in the treatment of GCs and to provide clinical staff with a reliable treatment regimen. In addition, through this study, it is believed that more and higher quality original studies will be designed and carried out to provide more accurate guidance for the treatment of GCs.The authors would like to thank the following people who either provided feedback on the protocol or supported the development of the methods: Jun Chen, Qifang Liu, Jun Xiong, Lunbin Lu, Genhua Tang, Siyuan Zhu, Zhiying Zhong, Xingchen Zhou, Han Guo, Zhijun Chen.All authors have read and approved the publication of the protocol.Conceptualization: Jun Chen, Jun Xiong.Data curation: Jun Chen, Lunbin Lu, Siyuan Zhu, Zhiying Zhong, Xingchen Zhou, Han Guo, Genhua Tang.Formal analysis: Jun Chen, Lunbin Lu.Investigation: Jun Xiong, Jun Chen.Methodology: Jun Chen, Siyuan Zhu, Lunbin Lu.Software: Genhua Tang, Zhiying Zhong.Supervision: Jun Xiong, Zhijun Chen, Xingchen Zhou.Writing \u2013 original draft: Jun Xiong, Jun Chen, Qifang Liu.Writing \u2013 review & editing: Jun Xiong, Zhijun Chen, Lunbin Lu, Siyuan Zhu."}
+{"text": "Objectives: The major role of interleukin (IL)-1 in the pathogenesis of hereditary recurrent fever syndromes favored the employment of targeted therapies modulating IL-1 signaling. However the best use of IL1 inhibitors in terms of dosage is difficult to define at present.Methods: In order to better understand the use of IL1 inhibitors in a real-life setting, our study assessed the dosage regimens of French patients with one of the four main hereditary recurrent fever syndromes , TNF receptor associated periodic syndrome (TRAPS), cryopyrin associated periodic fever (CAPS) and mevalonate kinase deficiency). The patients were retrieved retrospectively from the JIR cohort, an international platform gathering data of patients with pediatric inflammatory diseases.Results: Forty five patients of the JIR cohort with a hereditary recurrent fever syndrome had received at least once an IL1 inhibitor (anakinra or canakinumab). Of these, 43% received a lower dosage than the one suggested in the product recommendations, regardless of the type of the IL1 inhibitor. Especially patients with FMF and TRAPS seemed to need lower treatment regimens; in our cohort none of the FMF or TRAPS patients received an intensified dose of IL-inhibitor. On-demand treatment with a short half-life IL-1 inhibitor has also been used successfully for some patients with one of these two conditions The standard dose was given to 42% of the patients; whereas an intensified dose of IL-1 inhibitors was given to 15% of the patients . In our cohort each individual patient\u2019s need for treatment seemed highly variable, ranging from on demand treatment regimens to intensified dosage maintenance therapies depending on the activity and the severity of the underlying disease.Conclusion: IL-1 inhibitors are a good treatment option for patients with a hereditary recurrent fever syndrome, but the individual need of the dosage of IL-1 inhibitors to control the disease effectively seems highly variable. Severity, activity but also the type of the underlying disease, belong to the parameters underpinning the treat-to-target strategy implemented in an everyday life practice. Interleukin (IL)-1 is implicated in the pathogenesis of several systemic auto-inflammatory disorders and this recognition has favored the employment of targeted therapies modulating IL-1 signaling in a wide number of diseases . SeveralDespite the studies giving short or medium-term results, the use of IL-1 inhibitors on a long term and especially in real life may differ in terms of both intervals between the injections and dosage. Indeed, patients responding insufficiently to IL-1 inhibition, respond completely to a dose increase or shortening of the interval between the doses . ConversIn French tertiary care centers, IL-1 inhibitors have been used off-labeled in theses indications for several years . The anahttp://www.fondationres.org/fr/jircohorte - NTC02377245). For the purpose of the study, only patients from French centers (pediatric and adult) with complete history data and at least one completed follow-up visit were analyzed. Inclusion criteria to the study were all patients.1) with a monogenic autoinflammatory recurrent fever syndrome  according to the EUROFEVER/Printo classification criteria .2) who received during their follow-up at least one IL-1 inhibitor.Patients were identified from the JIR cohort, an international multicenter data repository granted by the Swiss-Children-Rheumatisms foundation, which aims to collect both retrospective and prospective information in a variety of juvenile onset systemic inflammatory disorders . Patients were enrolled after comprehensive information checking that they  were not opposed to the study and the storage of their personal data. The electronic case report form has been the object of an approval of the national commission for Data Protection and Liberties (CNIL).The primary objective of the study was to evaluate the consistence of dosing of IL1inhibitors in HRFs based on European Medicines Agency labeled recommendations.The secondary aims were 1) to analyze the reasons for discrepancies with the product recommendations and 2) to assess the overall safety profile of IL-1 inhibitors in HRFs.All the patients who received at least one IL1 inhibitor for colchicine resistant FMF, MKD, TRAPS and CAPS were assessed. Starting and ending date of the IL-1 inhibition were notified so that total exposure time for each IL-1 inhibitor, expressed in patient-years, could be calculated.To study the different dosage regimens, we considered the dosage of IL-1 inhibitor received at the last visit (or at the last visit before discontinuation of the studied IL-1 inhibitor). Patients were classified into three groups: group 1/lower than recommended dosage, group 2/standard dosage and group 3/intensified dosage. For anakinra, standard dose was defined as 100\u00a0mg/day (among adults) or 2 (\u00b10.5)\u00a0mg/kg/day (among children) . For canTo analyze the reasons for accordance or discrepancies of the different dosage regimens with the product recommendations, a descriptive analysis of the treatment modalities of the patients treated with IL-1 inhibitors was performed.Frequency and description of adverse events were retrieved according to the medDRA terminology. For each adverse event, investigators had to indicate the intensity among \u201cno effect\u201d, \u201cmild\u201d, \u201cmoderate\u201d, \u201csevere\u201d and \u201cvery severe,\u201d the seriousness with the necessity of an hospitalization or not, the relationship between the medication and the event among \u201cnot related,\u201d \u201cnot likely,\u201d \u201cpossible,\u201d \u201cprobable,\u201d and \u201cdefinitely\u201d and the consequence on the administration of the treatment among \u201cno action,\u201d \u201cdrug interrupted,\u201d \u201cdrug discontinued,\u201d \u201cdose reduced\u201d. Adverse events were expressed both as absolute number of events during the whole follow-up and as number of events/100 patients/year.Forty-five French patients who received at least once an IL-1 inhibitor, either anakinra or canakinumab or both, were identified in the JIR cohort and included for analysis. \u2022 Fifty percent of the patients (i.e 2 FMF and 3 TRAPS patients) treated with anakinra who received less than the recommended dose were treated with an on-demand regimen (anakinra administration only during flares), the other half received either a maintenance treatment by injections every other day instead of daily injections, or lower daily doses.\u2022 Administration modalities for canakinumab also varied: One CAPS and one FMF patient received an \u201con-demand\u201d regimen, i.e., an injection of canakinumab only if clinical and biological symptoms appeared. The other lower dose regimens involved patients with the new indication of canakinumab : they received less frequent injections than those stipulated in the SCP, varying from an injection every 10\u00a0weeks to every 6\u00a0weeks.The lower dosages in our cohort than the ones recommended in the summary of product characteristics (SPC) were explained by different treatment regimens:p = 0.55). No link could be established between the frequency of adverse events and the dosage of IL1 inhibitor received. Especially of the nine patients with a side effect considered as serious or very serious by the investigator, three received an intensified dosage regimen. No life-threatening adverse events were retrieved in our study.Concerning reported adverse eventsoccuring while on IL1 inhibitors , 6 led tp < 0.0001).The global drug retention rate was higher for canakinumab than anakinra : 33 out This study assessed the dosing regimen of IL-1 inhibitors in patients with a monogenic auto-inflammatory disease. During the study period, in France licensed use of IL-1 inhibitors was possible only in CAPS patients. Nevertheless the French healthcare organization enables physicians belonging to secondary or tertiary care centers for rare diseases to prescribe off labeled drugs and our study focused on these patients.Almost half of the patients received lower dosages of IL-1 inhibitors than the recommended standard dose. These lower dosage regimens concerned 60% of the patients with the more recent licensed indications of IL1-inhibitors: crFMF, TRAPS and MKD . EspeciaThe other main reason for lower dosages was an on-demand treatment strategy in FMF and TRAPS patients. An on-demand strategy was previously described only in 3 studies with anakinra . In a retuberculosis infection or reactivation, nor opportunistic infections were reported in our study. Our observations are comforting about the safety profile of IL1 inhibitors in HRFs and support the hypothesis that severe adverse events with IL1 inhibitors are preferentially related to the underlying diseases requiring IL1 inhibition and to the poor general clinical condition, rather than to an actual effect of IL-1 blockade  , but onlblockade .We show a far better drug retention for canakinumab than for anakinra, whereas side effects seemed equally frequent in both groups. Our hypothesis is that the ease of treatment may be the most important point for treatment persistence in patients. It is worth noting, that during the scheduled switch from anakinra to canakinumab, none of the attending physicians pointed out that anakinra was not sufficiently effective to justify changing the medication. Similarly, patients with on-demand anakinra therapy with inadequate disease control switched directly to canakinumab\u2013and not daily anakinra - maintenance therapy. These observations suggest that the ease of treatment is also a major argument guiding the choice of the drug for the prescribing physician.The major flaw of our study is that due to the retrospective design of our study; we were not able to retrieve a standardized disease activity score and consequently we were not able to link the disease activity of the patients to their treatment regimens. However we consider that we can infer the control of disease activity indirectly by assuming that the adaptations of therapies decided by the investigating physician were made because of criteria related to the severity and the control of the disease. The observed highly variable treatment regimens, ranging from on demand treatment regimens to intensified dosage maintenance therapies, reflects in our opinion that in daily life the investigating physicians adapts drug dosages as closely as possible to disease activity. This is all the more true since our study took place before the French marketing authorization for IL1-inhibitors in HRFs, at a time when dosages had not yet been standardized by the SCP.A second bias of our study concerns the heterogeneity of our sample, particularly concerning pathologies. However, this heterogeneity also highlighted that individual treatment needs are highly variable. Future studies should focus on identifying and refining the parameters underpinning the treat-to-target strategy practiced in HRFs.(1) IL-1 inhibitors are a good treatment option for patients with a hereditary recurrent fever syndrome.(2) The individual need of the dosage of IL-1 inhibitors to control the disease effectively seems highly variable, with about 45% of patients responding well to low dosages of IL-1 inhibitors.(3) On-demand treatment with a short half-life IL-1 inhibitor may be a treatment option for some selected patients with a recurrent hereditary fever syndrome.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by French Ethic Committee (CCTIRS). Patients were enrolled after comprehensive information checking that they  were not opposed to the study and the storage of their personal data.VH and SG-L were involved in the conception and design of the study. VH, SG-L, IK-P, AB, CG, GG, AC, AP, MH, and PP organized the data base. VH and MD analyzed the data. VH wrote the first draft of the manuscript. All authors contributed to the manuscript revision, read and approved the submitted version.No specific funding was received from any bodies in the public, commercial or not-for-profit sectors to carry out the work described in this article.VH, IK, GG, MH, and SG-L received personal fees and non-financial support from Novartis and SOBI; CG and AB received non-financial support from Novartis; AP received non-financial support from SOBI.The remaining 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."}
+{"text": "This study has investigated the immediate effect of induced hindlimb length difference on hindlimb lameness measured as differences in minimum (Pmin) and maximum (Pmax) pelvic heights in 16 horses trotting in a straight line and lungeing on both hard and soft surfaces with body-mounted inertial sensors. Hindlimb length differences were induced by applying an Easyboot Glue-on shoe to one hindlimb. Changes in Pmin and Pmax with induced hindlimb length difference were assessed with a two-way repeated-measures ANOVA with trial  and surface  as within-subject factors. Change in Pmin, indicating an impact-type lameness, in the hind limb with the elevation, was significant in both the straight line and while lunging on both hard and soft surfaces. Change in Pmax, indicating pushoff-type lameness, in the opposite, non-elevated hind limb, was significant when trotting in a straight line but not while lunging. Measurement of asymmetrical vertical movement of the pelvis is a common and accepted method to detect hindlimb lameness. The pelvis falls to a minimum height during, and rises to a maximum height after, stance, twice during one stride\u20135. In hoBut, it is reasonable to assume that not all horses will have equal hindlimb length, even if the horse is healthy and functioning normally. Hindlimb length difference can result from growth discrepancies of the hooves or long bones between the right and left limbs. These discrepancies may have been caused by injuries that are healed and no longer painful, or differences in functional demand from specialized training or repetitive movement, leading to the development of a type of \u201cleggedness\u201d similar to \u201chandedness\u201d in humans, 7. SuchDifferentiating lameness caused by hindlimb length difference or pain has important diagnostic and therapeutic ramifications. The effect of hindlimb length difference induced by hoof height elevation on equine hindlimb lameness has been initially studied by Vertz, et al. in 2018, where it was found that minimum and maximum pelvic height differences, as measures of hindlimb lameness, were significantly influenced in horses trotting in a straight line. However, in this study the effect of hindlimb length difference was only evaluated in horses trotting in a straight line on hard surfaces, limited conditions compared to common clinical evaluation [The purpose of this study was to investigate the effect of induced hindlimb length difference on differences in minimum and maximum pelvic heights in horses trotting in a straight line and lunging on both hard and soft surfaces. We expected that, after induction of hindlimb length difference, differences in minimum and maximum pelvic height would reflect simple differences in limb length, with higher minimum and maximum heights in the limb with increased length. This would result in a false measurement of impact type hindlimb lameness (differences in minimum pelvic height) in the limb with increased length, and of a false pushoff type hindlimb lameness (differences in maximum pelvic height) in the limb opposite the limb with increased length, under all conditions .Fifty horses used in an equestrian program and owned by a private university were evaluated for potential inclusion in this study. The William Woods Veterinary Ethics Committee governing the care of included horses approved use of horses in this study. All horses were being cared for and ridden daily by disciplined equestrian trainers and college students. Medical care was monitored and directed by a resident faculty equine veterinarian specialized in equine practice with over 20 years of experience evaluating lameness in horses (PS). Screening for enrollment in the study was initiated six months prior to the anticipated start date. Only horses that were not considered lame by simple subjective observation by the resident veterinarian, that did not measure with consistent hindlimb lameness, and that did not have existing tubera coxae height asymmetry while standing squarely were evaluated for enrollment. Throughout and after the study the horses continued to be used routinely in the equestrian program.Horses were instrumented with a body-mounted inertial sensor system (BMIS) consisting of a head, pelvic, and right forelimb sensors (Equinosis Q with Lameness Locator). The sensors on the head and pelvis measure vertical acceleration and convert to vertical position using an error-correcting double integration technique . The rigIn this study, only pelvic sensor measurements of hindlimb lameness were evaluated. For each stride, the minimum (Pmin) and maximum (Pmax) pelvic height differences between left and right stride halves were calculated . Pmin meA random number generator was used to determine if the control (before induction of hindlimb length difference) or treatment (after induction of hindlimb length difference) trials were collected first. The subsequent trials, treatment if the control was collected first, or control if treatment was collected first, were collected the same afternoon, or if not possible due to time constraints, the following day. Some trials were collected on separate days also to minimize the perceived effect of fatigue seen in some horses. Prior to collection, horses were lightly walked or lunged for 5\u201310 minutes. Hindlimb lameness was measured before and after induction of hindlimb length difference during a straight-line trot and lungeing in both directions on both hard (dirt) and soft (sand/fiber) surfaces. All horses had previously been trained to lunge and did so routinely as part of the equestrian program. Lungeing was performed in the same confined areas and by the same handler who had experience in both lungeing and working with the included horses. All horses were lunged in 8-m radius circles at a speed consistent with a working trot for the horses\u2019 discipline. For straight line trot trials, the horse was trotted 90 meters back and forth, twice. This resulted in the collection of data for at least 25 trotting strides. Data was collected for lungeing trials continuously for about 1 minute. This resulted in the collection of data for at least 40 trotting strides. Speed of movement before and after hindlimb elevation was controlled by measurement of stride rate, keeping stride rate for both before and after elevation trials within a +/- 0.2 strides per second.The Easyboot Glue-On shoe , with a ground surface thickness of 12.5 mm, was used to induce hindlimb length difference. The side of hindlimb length elevation was selected via a random number generator. The shoe was secured to the hind foot using Elastikon\u00ae 3-inch elastic tape. Nothing was applied to the other hindlimb.To estimate the amount of pelvic asymmetry caused by induced hindlimb length differences, pelvic height from the ground was measured on both the left and right sides, before and after induction of hindlimb length difference. This was measured (as performed in a previous study) as the vertical distance from the ground to the tuber coxae using a tape measure with units of +/- 0.1 cm. . During Sixteen adult  horses , out of the 50 screened were included in the study. Six of the horses performed as hunter/jumpers, 6 were western performance horses, and 4 performed as dressage horses. All excluded horses measured with more than weak evidence of mild hindlimb lameness. None of the horses were excluded for pre-existing tuber coxae height asymmetry. The elevation was applied to the left hindlimb in 10 horses and the right hindlimb in 6 horses.To maximize power for the small subject numbers in a specific group, we pre-processed the data as follows. Elevation of the left and right hindlimb were combined for straight line trotting by multiplying the difference in Pmin and Pmax between before and after hindlimb length elevation by -1. This results in positive changes in the lameness measure to indicate lameness in the elevated limb, and negative changes in the lameness measure to indicate lameness in the non-elevated limb. Lunging trials were combined into inside limb and outside limb elevation groups by multiplying all left lunge trials by -1. This results in positive changes in the lameness measure to indicate lameness in the inside limb and negative changes in the lameness measure to indicate lameness in the outside hindlimb. Changes in Pmin and Pmax with induced hindlimb length difference were assessed using a two-way repeated-measures ANOVA with trial  and surface  as within-subject factors. Diagnostic checks on the residuals were carried out to check normality and constant variance assumptions. Multiple comparisons were carried out by using the t-test statistic with Bonferroni adjusted p-values. Significance level was set at 0.05.We could not obtain accurate measurements of the distance between the ground and both tubera coxae before and after induction of hind limb length differences. Horses varied greatly in tendency to lean toward or away from the side of elevation. Also, in horses with large gluteal musculatures, the top of the tubera coxae was obscured, affecting the accuracy of the measurement. However, in all horses, it was apparent, despite these difficulties, that the height from ground to tubera coxae increased (approximately 50 mm) in the elevated limb, and was unchanged in the non-elevated limb.Results of hind limb elevation are summarized in This study investigated the effect of induced hindlimb length difference, and associated induction of pelvic asymmetry, on differences in minimum and maximum pelvic heights as measures of hindlimb lameness in horses trotting in a straight line and lungeing on both hard and soft surfaces. Difference in minimum pelvic height during stance, indicating an impact-type lameness, in the limb with the elevation, was consistently measured in both the straight line and while lungeing on both hard and soft surfaces. Difference in maximum pelvic height, indicating pushoff-type lameness in the opposite, non-elevated limb, was found only when the horse was trotting in a straight line.Our results, using 12.5 mm in hindlimb elevation, are in agreement with another study, where only straight line trotting was evaluated, and hindlimb elevations were greater (15 and 30 mm). In both studies elevation of a hindlimb caused the local minimum of pelvic height during stance of that hindlimb to be higher than before the elevation. However, in both studies, the magnitude of the mean change in Pmin was less the actual height of the elevation applied to the hindlimb. We measured a mean change in Pmin of about 6 mm with a shoe elevation of 12.5 mm, while Vertz et al measured a change in mean Pmin ranging from 2 mm to 6 mm for an elevation of 15 mm, and 8\u20139 mm for an elevation of 30 mm. Similarly, we measured a mean change in Pmax of about 3 mm with a shoe elevation of 12.5 mm, while Vertz et al. measured a change in mean Pmax ranging from 1 mm to 4 mm for elevations of 15 and 30 mm. It should be noted that Vertz, et al. used non-standardized values of Pmin and Pmax and the system we used standardizes Pmax and Pmin to the individual horse\u2019s total expected vertical pelvic movement, so that effects from very large or very small horses  do not skew combined results. Pmax and Pmin are standardized to the individual horse by dividing by the expected vertical displacement of the pelvis in that stride (the second harmonic after removing nonperiodic movement), and then multiplied by a constant to keep the measurement in units of mm. The slight differences in results between our study and Vertz, et al. could be because of this.Both studies show that the effect size for Pmax causing apparent pushoff lameness in the non-elevated limb is less than that for Pmin causing apparent impact lameness in the elevated limb. These results imply that the horse must be compensating by pushing off harder on the short limb compared to the limb that was elevated, so that the pelvis rises further upward after the foot of that limb leaves the ground. It also makes sense that Pmax would be less dependent than Pmin on absolute hindlimb length since Pmax is measured when neither hindlimb is in stance and Pmin is measured when one hindlimb is in stance.Pmax was not changed significantly at the lunge whether the hindlimb length elevation was on the outside or inside hindlimb on either soft or hard ground. When the horse is lungeing on hard ground, the amount of pelvic rise after pushoff of the inside hindlimb is normally less than that of the outside hindlimb\u201314, whicObjective measurement of vertical pelvic movement asymmetry to assess hindlimb lameness may be subject to error if there is a pre-existing reason for asymmetric pelvic height, like differences in hindlimb length. Results of this study suggest that, if the nature of hindlimb lameness is primarily lack of pushoff, i.e. with significant amplitude of Pmax, the cause is less likely due to a pre-existing limb length asymmetry, but to actual differences in pushoff force between the hindlimbs. However, if the nature of the hindlimb lameness is primarily lack of impact, i.e. with significant amplitude of Pmin, at least part of the cause may be due to a pre-existing hindlimb length difference.When using inertial sensors placed on midline to measure hindlimb lameness, if the measured hindlimb lameness is of a purely impact nature, one should check for pre-existing hindlimb length differences and pelvic asymmetry. This may be done by carefully standing the horse squarely on both hindlimbs and observing from the rear. Large amplitudes of pre-existing hindlimb length difference or vertical pelvic asymmetry should be taken into consideration when assessing Pmin. If the impact lameness is on the side of the higher hemi-pelvis, the true lameness may be less than measured. If the impact lameness is on the side of the lower hemi-pelvis, the true lameness due to pain may be higher than measured. Therefore, horses with pronounced pelvic asymmetry, may give the impression of a more severe lameness than actual, on the side of the higher hemi-pelvis, and of a less severe lameness than actual, on the side of the lower hemi-pelvis.In human studies, the effects of limb length discrepancy on ground reaction forces are inconsistent, with some indicating increased impact force on the shorter limb, 16. HowThe long-term effect of compensation for limb length differences and pelvic asymmetry may be detrimental to the horse. Standardbred Trotters with hindquarter asymmetry had poorer racing performance than those without hindquarter asymmetry. In humaThe sample size of this study was relatively small, so only large changes in Pmin and Pmax were likely to be found. Due to concern by both owners and trainers involved in this study of the possibility of induced limb length difference adversely affecting overall health and performance in these horses that were in active training and use, the long term effect of hindlimb length difference could not be studied. It is possible that short-term compensation methods used by the horses would wane over a longer period of time. The small sample size and our restriction to studying only immediate effects prevents us from making generalizations of the effects of hindlimb length difference arising naturally over longer periods of time.Horses with forelimb lameness will shift weight backward onto the contralateral hind limb during weight bearing, and they may pushoff less on the contralateral hind limb, affecting Pmax and Pmin. An existing forelimb lameness my cause a compensatory impact type ipsilateral hind limb lameness (causing a positive Pmin for a right forelimb lameness and a negative Pmin for a left forelimb lameness) and/or compensatory pushoff type contralateral hindlimb lameness (causing a negative Pmax for a right forelimb lameness and a negative Pmax for a right forelimb lameness). Although we did not control for or eliminate horses for possible use in the study by measuring for forelimb lameness, no horses with observable forelimb lameness were selected for use and no horses developed observable forelimb lameness during the study.In this study we measured asymmetric vertical pelvic movement aligned within the horse-centered global axes system  with sensors placed on the midline of the horse\u2019s body. We did not measure pelvic rotation that currently requires placing sensors off midline, on both sides of the body. It is possible that hindlimb length asymmetry does not create the same artefactual effect (a measured lameness caused only by limb length asymmetry) on measures of hindlimb lameness associated with pelvic rotation . Hip hike was not affected when elevation was placed on one hindlimb and horses were evaluated trotting in a straight line (8).,, the measure of pushoff-type hindlimb lameness, was affected, but to a lesser degree than Pmin, during straight line evaluation, but not during the lunge.This study has investigated the effect of induced hindlimb length difference on differences in minimum and maximum pelvic height measures of hindlimb lameness in horses trotting in a straight line and lungeing on both hard and soft surfaces. Pmin, the measure of impact-type hindlimb lameness, was consistently affected, increasing on the side of elevation in the straight and at the lunge. Pmax"}
+{"text": "A dysregulated metabolism is a hallmark of cancer. Once understood, tumor metabolic reprogramming can lead to targetable vulnerabilities, spurring the development of novel treatment strategies. Beyond the common observation that tumors rely heavily on glucose, building evidence indicates that a subset of tumors use lipids to maintain their proliferative or metastatic phenotype. This study developed an intra-vital microscopy method to quantify lipid uptake in breast cancer murine models using a fluorescently labeled palmitate molecule, Bodipy FL c16. This work highlights optical imaging\u2019s ability to both measure metabolic endpoints non-destructively and repeatedly, as well as inform small animal metabolic phenotyping beyond in vivo optical imaging of breast cancer alone.60), the mid-point in the plateau region, as a summary parameter to quantify Bodipy FL c16 fluorescence in subsequent experiments. Using our imaging platform, we observed a two- to four-fold decrease in fatty acid uptake in response to the downregulation of the MYC oncogene, consistent with findings from in vitro metabolic assays. In contrast, our imaging studies report an increase in fatty acid uptake with tumor aggressiveness , and uptake was significantly decreased after treatment with a fatty acid transport inhibitor, perphenazine, in both normal mammary pads and in the most aggressive 4T1 tumor model. Our approach fills an important gap between in vitro assays providing rich metabolic information at static time points and imaging approaches visualizing metabolism in whole organs at a reduced resolution.Targeting a tumor\u2019s metabolic dependencies is a clinically actionable therapeutic approach; however, identifying subtypes of tumors likely to respond remains difficult. The use of lipids as a nutrient source is of particular importance, especially in breast cancer. Imaging techniques offer the opportunity to quantify nutrient use in preclinical tumor models to guide development of new drugs that restrict uptake or utilization of these nutrients. We describe a fast and dynamic approach to image fatty acid uptake in vivo and demonstrate its relevance to study both tumor metabolic reprogramming directly, as well as the effectiveness of drugs targeting lipid metabolism. Specifically, we developed a quantitative optical approach to spatially and longitudinally map the kinetics of long-chain fatty acid uptake in in vivo murine models of breast cancer using a fluorescently labeled palmitate molecule, Bodipy FL c16. We chose intra-vital microscopy of mammary tumor windows to validate our approach in two orthotopic breast cancer models: a MYC-overexpressing, transgenic, triple-negative breast cancer (TNBC) model and a murine model of the 4T1 family. Following injection, Bodipy FL c16 fluorescence increased and reached its maximum after approximately 30 min, with the signal remaining stable during the 30\u201380 min post-injection period. We used the fluorescence at 60 min (Bodipy Cellular metabolism involves a vital network of pathways for homeostasis, growth, and survival. Alterations in these pathways are key features of a variety of diseases and conditions, such as diabetes, obesity, and cancer ,2,3. In The use of lipids as a nutrient source is of particular importance in breast cancer, as an increase in lipid droplet formation is associated with increased tumor aggressiveness ,10. AdipNovel methods to quantify the use of new energetic sources, including lipids, in preclinical models will aid in the development of drugs to restrict uptake or utilization of these nutrients. In vitro and ex vivo assays, like the Seahorse Assay and metabolomics, quantify multiple metabolic endpoints; however, both are restricted to single, static time points and sacrifice spatial information. In vivo imaging technologies have the potential to allow for repeated measurements of single or multiple metabolic endpoint(s) for dynamically studying metabolic reprogramming. Further, imaging-based techniques could be used for studying the effectiveness of metabolically targeted therapeutics within the tumor microenvironment.The ability to directly study fatty acid uptake within the context of an intact tumor-bearing animal is critical for evaluating novel therapeutics that antagonize lipid pathways or use lipids as an energy source. The most common in vivo metabolic imaging modality currently used for metabolic imaging is FDG-PET to measure glucose uptake with a radio-labeled glucose tracer . PreviouOptical imaging allows for the temporal mapping of fatty acid uptake, as seen in PET imaging, but at a high enough resolution for spatial mapping of preclinical tumors, providing complementary metabolic information to existing techniques. Additionally, optical approaches have the capacity to image several endogenous sources of contrast  and can Here, we adapted and rigorously validated the fluorescent palmitate analog, Bodipy FL c16, for in vivo imaging of fatty acid uptake. For this study, we specifically developed our injection strategy and summary time points using intra-vital microscopy of a mammary window breast tumor model so as to visualize local changes in fatty acid uptake in the precise orthotopic region where the tumor was implanted; however, our approach can be adapted to optical imaging using a variety of technologies that differ in terms of depth of penetration, resolution, and field of view. Two different endpoints were used in our study: a summary parameter for overall Bodipy FL c16 uptake and a heterogeneity parameter providing spatial information. We performed intravital Bodipy FL c16 imaging of two syngeneic breast cancer models: the MMTV-Tet-O-MYC conditional model of triple negative breast cancer (TNBC) (MTB-TOM)  and the Due to their destructive nature, existing technologies that measure long-chain fatty acid uptake within tumor tissues are often limited in their ability to temporally study a single tumor ,42. FluoFor intravital imaging, a 12 mm diameter titanium window was implanted over the tumor in the 4th mammary gland of each female mouse. The field of view within the window was excited at 488 \u00b1 5 nm and collected at 515 \u00b1 3.5 nm emission for fluorescence imaging C. To accPrevious studies found that tumors that overexpress the MYC oncogene, such as receptor triple-negative breast cancers (TNBC), have increased uptake of stable isotope-labeled fatty acids in a MTB-TOM model . We chos3; p < 0.05), and tumors\u2019 Bodipy FL c16 uptake was significantly decreased, ~3-fold less at peak (p < 0.05) compared to MYC-on tumors.60), the mid-point in the plateau region, as a summary parameter to quantify Bodipy FL c16 fluorescence in subsequent experiments. Thus, MYC-dependent uptake of long-chain fatty acids can be visualized with Bodipy FL c16 in accordance with previously reported stable isotope uptake studies in this model [To illustrate that the delivery kinetics of Bodipy FL c16 were independent of Bodipy FL c16 uptake level, each mammary window\u2019s kinetic curve in is model .Slc27a3 was the mRNA sequence in the family that decreased in expression, though not significantly . While it is possible that multiple mechanisms contribute to increased Bodipy FL c16 uptake in MYC-on tumors, which are beyond the scope of the present study, we have identified at least one FATP-family member, FATP3, whose expression is correlated to MYC in a model of TNBC.Long-chain fatty acid (LCFA) uptake and trafficking within cells can be mediated by multiple receptors and carriers. Therefore, the increased Bodipy FL c16 uptake observed in MYC-on tumors may be mediated by multiple effector molecules. To evaluate which effector molecules are important for fatty acid uptake in MYC-driven tumors, we characterized the effect of MYC expression on cell surface transport proteins which have been reported to mediate LCFA uptake . CD36 anficantly . Howevergression . FATP3 p60 fluorescence for MYC-off tumors compared to MYC-on tumors (p < 0.05) using a Wilcoxon signed-rank test . Additionally, probability density functions (PDFs), as shown in p < 0.05), signifying a decrease in the overall fluorescence (fatty acid uptake) in the tissue regardless of inter-tumor heterogeneity.With the importance of fatty acids in these MYC-on tumors established, we next explored how our method can longitudinally track metabolic changes in each animal following MYC downregulation. Longitudinal imaging was performed by imaging Bodipy FL c16 uptake in animals at baseline with MYC-on (+dox) and after MYC was turned off for four days (\u2212dox for 4 days) A. Backgrank test B. Notabl60 (TMRE fluorescence 60 min post-injection) images for MYC-on and MYC-off breast tumors (60 fluorescence was observed following four days of MYC downregulation (60 fluorescence shifted left in MYC-off (\u2212dox) tumors relative to MYC-on (+dox) tumors, as shown by the PDFs in LCFAs serve as an important carbon source for oxidative phosphorylation after being broken down through \u03b2-oxidation in MYC-on tumors ,57; thert tumors D. Roughlgulation E. AdditiWe next demonstrated that Bodipy FL c16 imaging can be applied to study in vivo efficacy of drugs that attenuate LCFA uptake. We tested whether perphenazine, a drug previously shown to inhibit LCFA uptake ,59, can p < 0.05). We also observed comparable results with two other cell lines from the same family (60 images of a 4T1 tumor with and without perphenazine treatment are shown in 60 in 4T1 tumors (p < 0.05). We observed comparable results with in vivo non-tumorous mammary tissue treated with and without perphenazine . We probed a recently published RNA-sequencing dataset and found that 4T1 cells have higher Myc mRNA when compared with 67NR cells [Myc expression, it was not significantly different between 67NR and 4T1 cells (p < 0.05). 4T1 tumors also showed increased heterogeneity compared to 4T07 tumors that trended towards significant (p = 0.057).The local range values for each pixel were plotted against its corresponding BodipyNR cells . While ST1 cells . These dOur and others\u2019 prior work indicate evidence of fatty acid dependencies in certain cancers ,64,65,66We tested our imaging approach in a MYC model, which has previously been shown to take up fatty acids from the circulation, validating the ability of our method to quantify fatty acid uptake in a regulated system. Bodipy FL c16 imaging, however, has the added advantage of permitting dynamic temporal imaging as MYC-driven tumors form and regress. The MTB-TOM model showcases two critically needed features for clinically relevant studies of fatty acid uptake: (1) longitudinal metabolite tracking in a single animal for intra-animal decreases in fatty acid uptake following oncogene downregulation, and (2) providing a link between fatty acid uptake and tumor aggressiveness.Longitudinal imaging of Bodipy FL c16 led to the finding that MYC-dependent tumors have decreased fatty acid uptake following MYC downregulation, which serves as a proof of concept for future testing of MYC-specific therapeutic interventions. This decreased uptake is also correlated with a visible reduction in tumor size . We linkIntravital Bodipy FL c16 imaging can also report on the activity of fatty-acid-uptake-targeted therapies, such as perphenazine, both in vivo and in vitro. While we do not know the exact mechanism, perphenazine likely targets the FATP family. FATP (1\u20136) have roles in fatty acid uptake and/or activation in cells, so our group sought to image their effects on Bodipy FL c16\u2032s uptake kinetics . Prior rMyc mRNA expression is higher in 4T1 cells in comparison with 67NR cells. This is consistent with previous findings that breast cancer patients with the highest Myc gene signature are most likely to have disease recurrence [Src significantly inhibited the metastatic burden of 4T1 tumors [Previous studies have implicated increases in both fatty acid uptake and \u03b2 oxidation in overall tumor aggressiveness and metastatic potential beyond MYC-inducible models ,69,70,71currence . Aside fcurrence . SRC exp1 tumors ,74. The 1 tumors ,76.Although 4T1 and 4T07 tumors have comparable Bodipy FL c16 fluorescence intensities, which are significantly higher than that of 67NR tumors, 4T1 tumors showed a greater degree of heterogeneity, measured by local range, compared to both 67NR and 4T07 tumors. Along with increased fatty acid metabolism reportedly correlated with increased metastatic and aggressive tumors, tumor metabolic heterogeneity has also been reported in the literature to correlate with increased tumor aggressiveness . PreviouThis work demonstrates that optical imaging of Bodipy FL c16 is important for lipid metabolism research. These studies are paramount to the successful translation of targeted metabolic therapies for TNBC and the plethora of other tumors that are dependent on fatty acids ,63,82. TIn addition to the disease models used here, this technology can be used in cancer pharmacology research to quantify metabolic changes in human tumors following therapies through the use of patient-derived xenograft (PDX) models and organoids for prognostication and to inform future treatment regimens, as both have shown to recapitulate metabolic features seen in patients ,88. In fOptical imaging is complementary to currently available metabolic imaging methods, including positron emission tomography (PET) as well as magnetic resonance spectral imaging (MRSI). Each of these imaging approaches measures key metabolic endpoints by providing short-term uptake kinetics of labeled metabolites with differing tradeoffs. PET imaging, though most prevalently used for imaging glucose uptake, can also image fatty acids  and mitoWe provide evidence supporting the use of fatty acid uptake with other validated metabolic imaging techniques (TMRE) to indicate effects on fatty acid oxidation in these tumors through the addition of mitochondrial membrane potential imaging . These r2)/2, where width represents the smallest axis and length the longest axis.All in vivo murine experiments were conducted according to the Duke University Institutional Animal Care and Use Committee (IACUC) (Protocol A072-18-03). All mice were housed in an on-site housing facility with ad libitum access to food and water with standard light/dark cycles. Tumor volumes were measured using calipers and calculated as Volume =   (Universn = 6 mice), 4T07 (n = 5 mice), or 67NR (n = 5 mice) cells suspended in sterile RPMI-1640  with no FBS or antibiotics using a 27G needle. Tumors were imaged at a volume of ~150 mm3.The 4T1 and 4T07 cells were acquired from the American Type Culture Collection, and the 67NR cells were generously provided by Dr. Fred Miller  through Dr. Inna Serganova and Dr. Jason Koucher . Cell lines were passaged every 2 days in Roswell Park Memorial Institute (RPMI)-1640 medium (L-glutamine) with 10% fetal bovine serum (FBS) and 1% antibiotics (Penicillin-Streptomycin). For in vivo injection, the abdomen of 6\u20138 week old female BALB/c mice (Charles River) was cleaned using 70% ethanol and the 4th right mammary gland was palpated and injected with 100\u2009\u03bcL  of 4T1  ,61. DataTumor tissue samples were flash-frozen in liquid nitrogen, and protein was extracted via a Dounce homogenizer in a cocktail of RIPA buffer  and proteinase  supplemented with phosphatase (Roche) inhibitor. Protein samples were resolved using 4\u201312% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels  and transferred to nitrocellulose membranes . To probe for FATP3  and c-MYC (MYC) , membranes were first incubated in primary antibody overnight on a 4 \u00b0C shaker, then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. To probe for \u03b2-actin (actin), membranes were incubated for one hour at room temperature in horseradish peroxidase (HRP)-conjugated antibody . Signals were visualized with enhanced chemiluminescence (ECL) , and chemiluminescence was acquired via Bio-Rad ChemiDoc XRS+. Unsaturated band intensities were quantified using Fiji software.Using a previously established procedure, titanium window chambers with 12 mm diameter, No. 2 glass coverslips, were surgically implanted over the 4th right mammary gland of 8\u201312 week old female BALB/c (Charles River) .For in vitro imaging, Bodipy FL c16  was diluted in sterile RPMI-1640 (Corning) to a final concentration of 1 \u00b5M and incubated in each cell plate for 30 min. Next, cells were washed and imaged immediately with a standard confocal microscope . A total of 9 representative fields of view across 3 independent plates per cell line were imaged, and each cell line was measured on at least two different days.For in vivo administration, Bodipy FL c16 was diluted to a final concentration of 200 \u00b5M in dimethyl sulfoxide (DMSO). This concentration was titrated to achieve a final tissue-level concentration ) below levels where self-quenching has been observed  and simiv/v) in room air in an induction chamber. The animal was transferred to a heated (to maintain core body temperature) microscope stage where a background image (laser on) and dark image (laser off) were acquired prior to probe administration to account for signals from sources other than the fluorescence probes. Each animal received 100 \u00b5L of 200 \u00b5M Bodipy FL c16 in DMSO or 75 \u00b5M of TMRE in sterile PBS via tail vein injection and fluorescence images were acquired for 80 min. An exposure time of 5 s was used for both Bodipy FL c16 and TMRE images. To account for daily light source variation, all images were background subtracted, beam shape corrected, and calibrated according to a Rhodamine B standard imaged at each imaging session prior to image analysis. Each mouse was imaged only once. The only exception is with our genetically engineered mouse (GEM) model to track changes in metabolism on dox (MYC-on) compared to no dox (MYC-off), where each mouse was imaged twice, four days apart.All mice were fasted for 4 h (water provided) prior to imaging to ensure a normalized metabolic rate for each animal . FollowiOur previously reported microscope  was usedThis system uses a Nikon CFI E Plan Achromat 4\u00d7 objective  for all imaging. This creates a single frame field of view of 2.1 \u00d7 1.6 mm and a lateral resolution of 2.2 \u00b5m, as measured using a 1951 USAF resolution target . This miFor in vitro inhibition experiments with perphenazine, 4T1 cells were treated with 80 \u00b5M perphenazine  dissolved in sterile RPMI-1640 (Corning) for 2 h prior to in vitro staining and imaging according to the methods above. A total of 9 representative fields of view across 3 independent plates per condition were imaged, and each condition was measured on at least two different days. To ensure doxycycline does not interfere with Bodipy FL c16 uptake, 4T1 cells were treated with 10 ng/mL doxycycline dissolved in sterile RPMI-1640  or PBS for 2 h prior to staining and imaged according to the protocol described above. A total of 9 representative fields of view across 3 independent plates per condition were imaged.n = 3 mice) or non-tumor (n = 3 mice) animals also received topical treatment with 80 \u00b5M perphenazine dissolved in sterile PBS. Following isoflurane anesthesia, the mammary window chamber was removed and 100 \u03bcL of perphenazine was applied to the exposed tissue. Following 2 h of incubation, sterile PBS was used to wash the window, the coverslip was returned to the tissue, and imaging began immediately according to the methods above.Separate cohorts of 4T1-bearing . Prior to further analysis, each image collected first underwent background and dark noise subtraction by removing the average value of an image collected with the laser source off (dark noise) and removing the average signal imaged prior to fluorescent probe injection (background). Additionally, due to the non-uniformity of illumination of a Gaussian light-source, each image was divided by an image of a uniform phantom to correct for the beam shape. Finally, each image was calibrated using a Rhodamine B standard solution imaged during each imaging session. These methods accounted for autofluorescence, non-uniform illumination, and day-to-day system variations, respectively. The resulting images were used for all statistical comparisons and images displayed in this study. The average intensity values at each imaging time point (0\u201380 min) were calculated to generate a time course kinetic curve. Comparisons of these curves were performed using a two-way analysis of variance (ANOVA). Additionally, comparison of average intensities using our summary parameter, BodipyAs the growing literature implicates fat as a key carbon source for many cancers, there is a need for a validated method to non-invasively image fatty acid uptake in vivo. In breast cancer alone, for example, exogenous fatty acid uptake and oxidation have been shown to be increased in primary TNBC , followi"}
+{"text": "Dalea purpurea Vent.), a native legume forage, with native cool-season grasses on the in vitro fermentation and in situ digestibility of mixed forages.Incorporation of legume species into native North American pastures is considered an effective method to increase native pasture productivity and improve the nutritive value of forage. This study evaluated the effects of inclusion of purple prairie clover , in vitro DM disappearance (IVDMD), total volatile fatty acids (VFA) and ammonia-N production. Mixtures of forages harvested when the PPC reached the FL stage and 50:50 mixture of forages harvested at VEG, FL and SP stages were incubated in the rumen of three heifers for 0, 2, 6, 12, 24, 48, 72 and 96\u2009h to determine in situ degradabilities of DM, neutral detergent fibre (aNDF) and crude protein (CP).Whole plant PPC and mixtures of cool-season grasses were harvested when the PPC reached the vegetative (VEG), full flower (FL) and seedpod (SP) stages, and were combined in ratios (DM basis) of 0:100, 25:75, 50:50, 75:25 and 100:0 at each maturity. P\u2009<\u20090.01), while CP decreased (P\u2009<\u20090.001) as PPC matured. Concentrations of extractable condensed tannins in PPC ranked as FL\u2009>\u2009VEG\u2009>\u2009SP (P\u00a0<\u20090.05). Regardless of PPC proportions in the mixture, GP decreased (P\u00a0<\u20090.05) with increasing PPC maturity. Increasing PPC proportions linearly increased (P\u2009<\u20090.001) GP, IVDMD and total VFA at VEG, but linearly decreased (P\u2009<\u20090.001) them at SP. Irrespective of PPC maturity, ammonia-N production linearly increased (P\u2009<\u20090.01) with increasing proportions of PPC and the concentration was higher (P\u2009<\u20090.05) at VEG than at FL and SP stages. Increasing proportion of PPC at either maturity linearly increased (P\u2009<\u20090.001) molar percentage of acetate (A) and branched-chain VFA, but linearly decreased (P\u2009<\u20090.001) molar percentage of propionate (P), resulting in a linearly increase (P\u2009<\u20090.001) in the A:P ratio. Increasing FL PPC in the mixture linearly and quadratically (P\u2009<\u20090.01) increased a (soluble fraction), but linearly and quadratically decreased (P\u2009<\u20090.01) b  for DM and aNDF, resulting in linear (P\u2009<\u20090.05) and quadratic (P\u2009<\u20090.01) increases in DM and aNDF maximum potential degradabilities (a\u00a0+\u00a0b). Effective degradabilities of DM and aNDF were also linearly and quadratically increased (P\u2009<\u20090.05), and CP was quadratically increased (P\u2009<\u20090.05) with increasing FL PPC, with the greatest effective degradability being observed with ratios between 50:50 and 75:25. Ruminal maximum potential degradabilities of DM and aNDF decreased (P\u2009<\u20090.001) as the forage matured. Effective degradability of DM ranked as VEG\u2009>\u2009FL\u2009>\u2009SP (P\u2009<\u20090.001), whereas the effective degradability of aNDF was similar between VEG and FL and both were greater (P\u2009<\u20090.01) than SP.Contents of aNDF and ADF increased (Inclusion of vegetative PPC in a mixed forage diet resulted in the greatest digestibility and incorporation of PPC before seedpod stage with native grasses had a positive effect on ruminal fermentation. Effects of PPC on ruminal digestion depend on both the stage of maturity and its proportion in mixed legume-grass pastures. Pastures containing 50% of PPC in full flower stage would likely provide the greatest quality diet to grazing ruminants subject to potential animal selectivity. Grasses in native pasture are usually the principal forage source in the prairie region of North America during late season grazing. However, nutritive value of grasses rapidly declines in the latter half of the grazing season . InclusiDalea purpurea Vent.) is a perennial native legume that is well adapted to the North American prairies and has higher palatability and digestibility than other native legumes such as false indigo (Amorpha fruticosa L.), blue wild indigo  R. Br.) and wild senna  Irwin & Barneby) \u00a0ek)\u00a0L\u2212, which was set at 0.02 for aNDF and 0.05 for both DM and CP -grass (timothy (Phleum pratense L.) and smooth meadow grass (Poa pratensis L.) mixed forages [in vitro fermentation as a result of mixing a tropical grass  and a temperate legume  but not between peanut vines (Arachis pintoi), sainfoin (Onobrychis viciifolia) and grasses of axonopus and tall fescue (Festuca arundinacea).The reduction in the ruminal fermentation of PPC and cool-season grasses from vegetative to seedpod stages was reflected in the increase in ADF content of the two forages. Negative effect of ADF content on forage digestion has been demonstrated in literature . Interes forages . A quadr forages  reportedin vitro fermentation. Ammonia-N accumulation in closed in vitro system is the net result of ammonia from the degradation of dietary protein and utilization by microbes for microbial protein synthesis. The linearly-increased ammonia-N accumulation with increasing PPC at all maturities is a reflection of the increasing protein concentration in the mixture. Whether microbial protein synthesis (ammonia-N utilization) was affected by the inclusion of PPC was not determined in this study. Nevertheless, Jin et al. [in vitro experiment. Dal Pizzol et al. [Crude protein content in PPC was higher than that in cool-season grasses throughout the growing season. This resulted in an increase in the protein concentrations of the PPC-grass mixtures and the subsequent increase in ammonia-N concentration during n et al.  found thn et al. . Decreasn et al. , 11, 13.l et al.  also repl et al. , 37. Becin vitro and in vivo studies [Prevotella bryantii [Ruminobacter amylophilus [The result that increasing PPC proportion in the PPC-grass mixture increased acetate: propionate ratio by increasing acetate and decreasing propionate was consistent with Jin et al. . The var studies , 11, 13.bryantii  or Ruminlophilus , becauselophilus .in vitro and in situ studies showed PPC CT at concentrations up to 82\u2009g/kg DM had minimal impact on ruminal feed digestion [It is generally regarded that the nutritional role of CT in ruminant nutrition depends on their dietary concentrations and chemical composition , 42, 43.igestion , 12. In igestion . Huang eigestion  found thPurple prairie clover contained higher protein than cool-season grasses throughout the growing season and therefore the incorporation of PPC into cool-season grasses would increase the protein content of forage in rehabilitated native pasture. However, the improvement in nutritive value of the forage by the incorporation of PPC into native pasture depends on the PPC growth stage, with greatest benefit being obtained at the vegetative stage followed by full flower and seedpod stages. Considering the faster decline of nutrient digestion of PPC over the growth season than cool-season grasses and balanced by N content of the two types of forages, it seems that about 50% of PPC in the PPC-grass mixed forage would provide most benefit for the purpose of extending the grazing season. These results need to be confirmed with field trials to better understand competition ability between PPC and cool season-grasses."}
+{"text": "BJPsych Open concerning coronavirus disease 2019 (COVID-19) and healthcare. We report nine papers that cover concepts and epidemiology relating to the public and patients. We review 11 papers about the impact of COVID-19 on healthcare services and their staff in 15 countries. Two papers consider the psychosocial impact on staff working in mental health services in the UK. Most papers report cross-sectional analyses of data collected from convenience samples by self-reported surveys conducted at single times. They have limitations of generalisability, do not enable conclusions about diagnosis or causality, and many are likely to have attendant bias and noise. BJPsych Open published these papers to meet requirements for early indications of the mental health impact of COVID-19 on the public and on healthcare staff. They claim high prevalence of symptoms of anxiety, depression and post-traumatic stress. We contrast these findings with selected reports of studies with different methodologies published elsewhere. We emphasise the need for longitudinal clinical studies with refined sampling and methodological rigour. We identify several longitudinal research programmes; two in this series. We advocate tuning advice offered about caring for the public and healthcare staff to the realities of their circumstances and their perceptions of need in the context of findings from further longitudinal studies. We draw attention to the importance of the social, relationship and environmental circumstances of the public and healthcare staff in order to understand their distress and their risks of developing mental health disorders.This review covers the thematic series of 22 papers selected from among manuscripts published by  BJPsych Open has published during the pandemic. We draw them together in this thematic series to illustrate the huge volume of literature that has been presented to many journals in the last 18 months. This series leads to several major themes and subthemes on which we comment.This narrative review overviews a selection of 22 papers from among those concerning coronavirus disease 2019 (COVID-19) and healthcare that 1 To do otherwise \u2018\u2026 prevents us from understanding whether the effect of the pandemic on their mental health is different to other key workers or the general population\u2019.1 Thus, this review and our thematic series begins with illustrative papers about the general population.Although initially planning to focus this review on the experiences and the mental health impact of COVID-19 on staff working in healthcare services, we adjusted that aim because we agree with Lamb et al, that the experiences facing healthcare staff should be seen against the backdrop of the impact on wider populations.BJPsych Open is receiving papers that cover a wider array of practical matters and methods. Our selection reflects a diversity of topics. We draw attention to the strengths and weaknesses of the studies conducted early in the pandemic. Importantly, these considerations lead us to emphasise the need for longitudinal studies; without them, it is difficult to adjudge their interpretation, and the severity, duration and trajectories of the impact on mental health. Advised by research on other emergencies, major incidents, conflict and outbreaks of high-consequence infectious diseases, we recognise that the effects on the mental health of the public and professionals may be delayed, develop insidiously, take time to appear and may be protracted.2 Jordan et al document the impact on people's health of 9/11, and we recognise the poignancy, disability and sorrow that continues to attend that event 20 years later.2 It reminds us that the UK National Health Service (NHS) and all healthcare services need to anticipate and be prepared for potential increased mental health demands that stem from COVID-19 over an extended time period.Many of the papers in this thematic series focus on data collected in the early months of the pandemic, although, as time has progressed, Our opinion is that people's initial responses to such life-affecting events as this pandemic are very likely to represent their distress rather than mental health disorders. We require well-designed, appropriately powered and carefully executed clinical, longitudinal studies of representative samples constructed by, for example, probability sampling, to allow us to determine the nature and extent of the impact on people's mental health. Most of the research reported in the thematic series, including the mixed-methods and qualitative studies, used convenience samples. The quantitative studies used self-reported online screening questionnaires and/or psychometric scales that had been created for other research and validated previously. They provide snapshots of the impact and are illustrative of the papers that have appeared in the literature to date. In addition to the main findings, each paper presents some fascinating additional information that may have been useful in addressing the potential implications for the public and the health and social care workforce during the COVID-19 pandemic waves that have followed and in enabling the academic community to decide which risk and protective factors to research in the future. We look forward to adding papers to this selection as stronger research emerges.3 These developments came as the four governments in the UK were reducing the legal enforcement of measures that were designed to reduce transmission of the virus. The omicron variant of SARS-CoV-2 arrived in the UK in November 2021; by mid-December, it had replaced delta as the dominant variant in London and it became dominant across the UK before the end of 2021. The UK's governments reintroduced measures to try to reduce transmission of the virus. The policy is that a reasonable level of personal and public protection turns on public administration of booster vaccines against a very short deadline. The spread of omicron may well curtail the third wave and usher in a fourth due to the enormously high transmissibility of the mutated virus and its greater resistance to vaccines. The measures are causing even greater pressure on the NHS and its staff.We began assembling this series in April 2021 when the sheer level of fatigue of staff working in healthcare services in the UK was evident. We finished collating this series in October 2021. In the summer of 2021, pressure on staff working in the NHS in the UK started to settle somewhat although the impact of what staff had been through continued to ramify. In the UK in autumn 2021, and despite the fortitude of healthcare staff, pressures on them rose substantially again as a consequence of a virulent third wave caused by transmission of the delta variant of the SARS-CoV-2 virus; the mounting backlog of undelivered care; shortages of staff; and consequent continuing very high demands on services.There have been similar developments in many countries and the world situation remains precarious. With each additional variant, wave and potential restrictions, there may be varied psychosocial responses and the possibility of cumulative processes. Although longitudinal studies will best inform us about what to expect, we hope that insights gained from the initial waves and articles in this narrative review will help healthcare services and healthcarers to navigate future pandemics.4 Several papers presented online at the Trauma Care Conference in the UK in September 2021 made this point. They stir enormous gratitude and respect for the staff. We echo the sentiments of the Chief Medical Adviser to the UK Government in his presentation to the Royal College of Psychiatrists International Congress on 23 June 2021 when he recognised the enormous efforts and achievements of the staff working in healthcare services.These experiences raise huge concerns about the impact of working in such challenging and risky circumstances on the mental and physical health of staff.Many people have registered grave concerns about the impact of lockdown, quarantine and isolation on families and adherence of the public to the regulations whether or not people had contracted COVID-19. We include in the series three papers based on differing methodologies to draw out some potentially positive and challenging aspects of the ways in which the pandemic is managed.n\u00a0=\u00a0185) and the UK (n\u00a0=\u00a0200) of whom 70% were working exclusively from home.5 \u2018Almost half reported a reduction in income, [and] most children were home taught\u2019.5 Importantly, although the cross-sectional sampling methods reported in this paper impose limitations, a single qualitative question enabled the authors to identify that nearly 90% of participants reported elements of post-traumatic growth despite their experiencing considerable adversity.First, Stallard et al report a mixed-methods study in the first wave of 385 caregivers, mainly parents, in two European countries, Portugal  were recruited by repeated probability sampling to estimate prevalence of mental disorders and suicidal ideation across 2020. The authors carried out repeated cross-sectional analyses. Knudsen et al's paper supports caution about a tidal wave of impact on the mental health of the population.18 The authors showed that levels of mental disorders, suicidal ideation and suicide deaths were stable in the first 6 months of the pandemic and comparable with findings before the pandemic. Appleby also expressed caution based on reports from seven countries of national or state-level suicide data pointing out the suicide rates had not risen by spring 2021.19It is important to identify several longitudinal studies. First, is a paper that reports research on participants in the Tr\u00f8ndelag Health Study in Trondheim, Norway. Participants  to assess mental health in 19 763 adults. This research showed that most people remained resilient, or their mental health returned to pre-pandemic levels after a deterioration in the population mean in April 2020. \u2018One smaller group of people had sustained elevated scores (4.1% of the cohort) and another group (7.0% of the cohort) had little deterioration \u2026\u2019 but showed a steady decline in mental health over time. This paper also addresses predictors of deterioration of this nature and coming from an ethnic minority background, pre-existing mental illness, financial adversity and SARS-CoV-2 infection emerged as risk factors, as they have in other research. Pierce et al remind us of research on trajectories of psychosocial impact after major incidents that has documented a number of patterns of change over time of people's experiences or symptoms of stress and distress.24 The two most common patterns or longitudinal \u2018trajectories\u2019 are: (a) mild\u2013stable levels of distress; and (b) decreasing symptoms from moderate to mild over months and years. Smaller proportions of people affected by major incidents may show: (c) deteriorating patterns of worsening stress; or (d) high-stable levels of stress. These last two groups appear more at risk of psychopathology.Another paper considers the longitudinal trajectories of the public's mental health responses to the COVID pandemic in the UK.28 Both these papers are based on self-reported symptoms measured using screening questionnaires given to cross-sectional convenience samples. The response rates were either not stated or low.We include in this thematic series two papers that report public prevalence data for possible mental health disorders.25 In the first, the authors used random stratified sampling to construct an internet panel and the K6 to screen for \u2018serious mental illness\u2019. The response rate for study 1 was 37% and it found 16.6% of the participants who reported COVID-19-related social life or occupationally stressful events to have elevated levels of symptoms of serious mental illness. The second study was intended to replicate the findings from the first. The authors report an elevated risk of serious mental illness and probable ICD-11 adjustment disorder among people who say they have had stressful events related to COVID-19. Both studies showed stratified increased risk based on number of stressful events. This is an interesting finding because it resonates with the \u2018cumulative risk model\u2019. This model is the dominant thesis regarding children stating that \u2018\u2026 long-term adverse outcomes are better predicted by the total number, rather than the specific nature of environmental risk exposures\u2019 to \u2018\u2026 different adverse experiences and events\u2019 early in childhood.27The first, by Ben-Ezra et al, reports two studies of 1293 and 1073 UK participants in the early weeks of the pandemic.28 Participants were recruited through television webpages. This project identified escalating effect sizes with increased severity of COVID-19 and degree of treatment.In the second paper, Chamberlain et al report a study that examined, in May 2020, post-traumatic stress disorder (PTSD) symptoms in 13 049 people in the UK aged 16 or older who self-reported suspected or confirmed COVID-19, predominantly from the UK general population.29 Having COVID-19 was operationally defined as patients having \u2018\u2026 a positive antigen test for SARS-CoV-2 [real-time polymerase chain reaction test] taken during the patient's current period of hospital admission\u2019. All were in-patients in hospitals in Doha, Qatar who were referred to a consultation-liaison team. The authors report that a psychiatric diagnosis was made in 49 cases and that \u2018\u2026 a third of our sample had a past psychiatric history, and of these nearly half had a psychotic disorder or bipolar I disorder \u2026\u2019. The authors say that this \u2018\u2026 suggests that those with prior mental health problems are particularly vulnerable to develop further mental health problems during the pandemic\u2019.Research by Iqbal et al aimed to ascertain the psychiatric morbidity associated with SARS-CoV-2 infection by a retrospective study of case notes of 50 consecutive patients for whom psychiatric diagnoses reflected clinical judgement.30 The authors report that 138 of 176 patients did not comply with the measures and that people who had diagnoses of psychotic, personality and substance use disorders were less adherent than those with other disorders. Their adherence improved when they were given direct instructions. We recognise that there are many potential weaknesses in studies that use case notes to glean data retrospectively but this one shows the importance of giving patients clear instructions about protecting themselves and other people.Another retrospective case-notes study, by Williams et al, reports patients\u2019 adherence with measures taken by an acute psychiatric in-patient ward in London to control spread of SARS-CoV-2 among patients.31 Paterson describes a spectrum of COVID-19 neurology,32 Baig documents deleterious outcomes in \u2018long-haulers\u2019 who have a chronic COVID syndrome as a result of SARS-CoV-2,33 and Nalbandian et al, provide an account of persistent and prolonged effects after acute COVID-19.34 Hypotheses for understanding the origins, and treatments for these persisting effects of COVID-19 are presently emerging. One important observation is that the occurrence of long-haul problems is not necessarily related to the severity of COVID-19 experienced by patients.35It has also become clear that many people, including healthcare staff, have developed sustained healthcare problems of a broad nature that stem from embolic and other phenomena that have been caused by the SARS-CoV-2 virus and the immune responses to it. The National Institute for Health Research in England is publishing reviews of \u2018long COVID\u2019.1 By the spring of 2021, staff in the NHS in the UK were shattered, whether or not they had patient-facing jobs, although staff in more intense and senior management roles were especially so. This is likely to be similar in healthcare systems across the world. There can be no doubt about the degrees of stress and distress that staff have experienced and are experiencing. Murray et al offer a review of how the demands have been experienced by healthcare staff in the UK.22 They indicate that distress may result from primary stressors i.e. those sources of stress that are inherent in the pandemic including the risks to their own health and their fear of transmitting the virus to patients, relatives and colleagues. Also, we recognise that some health and social carers\u2019 experiences might result from the COVID-19 disease processes that can cause psychiatric symptoms.As Lamb et al noted in 2020, \u2018Anyone working in the health service at present has likely noticed \u2026 a proliferation of surveys on health-care workers.\u20193 In mid-December 2021, healthcare staff were moving from tackling the third wave in the UK, to dealing with demands caused by the omicron variant. Measures to slow the new variant's transmission have been introduced to find time to deliver a very demanding plan to offer booster vaccinations to the whole of the UK's adult population in a very short timescale. As we have observed, the demands on healthcare staff have been enormous. But now there are even greater pressures and NHS England declared a new Level 4 national incident and suspended some routine work in the light of the activity required by the vaccination programme and preparations for the potential significant increase in COVID-19 cases.From spring to autumn 2021, levels of fatigue among UK healthcare staff were almost palpable.During autumn 2021, there were proportionately fewer hospital admissions in the UK, consequent on the country's vaccination status. As we enter a fourth wave, the circumstances regarding hospital admissions and deaths as a result of the omicron variant are uncertain. This raises huge concern about the public, healthcare services but also about the impact on staff working in such challenging and risky circumstances for their mental and physical health, while also stirring enormous gratitude and respect for their selfless efforts.22 These claims have been experienced as hurtful by healthcare staff who have put themselves and their families at risk and have worked and are working long and hard to care for the public. Some have contracted COVID-19, no small number of staff has died, and some have contracted Long COVID.At times, conspiracy theorists have suggested that the extent of the pandemic has been exaggerated and, even, that its existence is a hoax.22 describe how the distress experienced by so many staff may also arise from secondary stressors. They are defined in Williams et al as: \u20181. Social factors and people's life circumstances  that exist prior to, but impact them during the major incident, emergency, disaster, conflict, or disease outbreak; and/or 2. Societal responses to the major incident or emergency\u2019.11 They may exacerbate primary stressors. They include matters that may be secondary to coping with the existence of COVID-19 and its ramifications, for example, telecommuting and remote education with decreased social contact. Although the secondary stressors might appear to be more trivial, our experiences show that some concerning longer-term matters such as access to showers, hot food and parking spaces have had a huge impact on staff during the pandemic.22 But chief of all these concerns among staff has been reliable access to personal protective equipment (PPE) that meets the standards set by the World Health Organization.Murray et al,36 Murray et al observed both the support mobilisation and support deterioration pathways among the public and staff working in the NHS during the first wave.22In summary, Murray et al draw attention to the importance of understanding that COVID-19 is having an impact on an NHS in the UK that was already chronically stressed, and this raises the importance of understanding the balance of primary and secondary stressors that staff faced and continue to face, and finding effective ways to support them. Furthermore, previous research on other types of major incident and emergencies has identified two broad pathways in which groups of people, first, mobilise support although, later, that support deteriorates.37 They say that these actions led to healthcare staff being exposed to extremely stressful situations and the authors projected \u2018\u2026 a rebound effect on mental health problems \u2026 in the medium and long term \u2026\u2019. They called for preventive approaches, use of telepsychiatry and evaluation of altered ways of providing mental healthcare. We have seen each of these proposals in action in the past 18 months.Our thematic series includes a personal account of the opening 6 months of the pandemic as it affected services and their staff in Spain. Pacchiarotti et al, describe how Spain reconfigured its hospitals and redeployed healthcare professionals to manage demand early in the pandemic.38 Thus, the potential for staff to experience moral distress and moral injury have been widely discussed in the UK though the pandemic. Murray et al introduce both moral distress and moral injury.22 Rimmer summarises claims made by the British Medical Association after its survey of doctors conducted in March and April 2021.39 She says that around half of responders had heard of moral distress and moral injury and 78% of responders (around 1800 doctors) said that moral distress resonated with their experiences at work whereas 51% said the same for moral injury. Doctors who worked only with patients who had COVID-19 were more likely to give these responses as were doctors from ethnic minority backgrounds compared with doctors who are White.An important matter concerns the risks to staff who address the ethical dilemma of choosing who receives services when healthcare systems are overwhelmed. Triage and triage-like decision-making remains with people who make those decisions for a substantial time afterwards.41 and these concerns continued through the second wave in the UK. In March 2021, the Royal College of Nursing published an independent report that found that the UK's guidelines for ventilating healthcare premises and face protection had not been updated to reflect evidence about airborne transmission of SARS-CoV-2.42Two papers in the thematic series reinforce the psychosocial importance of PPE availability with associated workplace training,43 They found that the organisational measures that best decrease the risk of adverse outcomes include: positive feedback to staff; the faith of staff in local infection control procedures; providing protective gear; effective preparation; and training.Early in the pandemic, Kisely et al, reported a systematic review of the psychological effects of what are now known as high-consequence infectious diseases (HCIDs) on healthcare workers over the past 20 years.49 We identify a mix of primary and secondary stressors emerging from these papers as associated with participants\u2019 experiences of distress and symptoms of mental health disorders. Seven of the papers in the thematic series (dates of sampling in parenthesis) report findings from a single country; two papers are from China ; three from the UK ; one from Ethiopia (March 202047); and one from Italy (March 202048). The eighth paper reports on healthcare staff in India, Indonesia, Malaysia, Singapore and Vietnam (April to June 202049). These papers offer differing but helpful snapshots of the mental health of staff in the first wave of the pandemic and all report high levels of psychiatric symptoms.The thematic series includes eight papers that describe surveys, mainly online, of the impact of the pandemic on healthcare staff conducted in a number of countries using self-administered screening tools with convenience samples.40 The authors found high rates of symptoms of depression (42%), anxiety (29%) and insomnia (34%) in a total sample of 2126 participants. \u2018Regardless of whether or not they had direct contact with patients with COVID-19, obstetricians and midwives were more likely to report mild and moderate depression and anxiety during the COVID-19 pandemic when compared with before the pandemic\u2019. Higher rates of symptoms of depression and insomnia were reported by staff who had direct contact with patients with COVID-19. \u2018Those who had sufficient protective equipment or training were less likely to report depression, anxiety and insomnia than those who did not\u2019.40In the first paper from China, Liu et al report mental health symptoms experienced by obstetricians and midwives who were not infected as they treated hospital in-patients who had COVID-19.44 There were 102 participants,  who stayed in the fever clinic during their rotation, and adjustments were made to their working conditions to help them adapt to the work. Staff received semi-structured interviews from staff of a hotline support service and the records were coded by scenario analysis and some answers were converted to binary variables. The total score on the Sources of Stress correlated moderately with the IES-R score. The top four sources of distress were worry about: the health of one's family or others; the spread of the virus; changes in work; and one's own health.Hong et al report a cross-sectional study of the psychological impact of the COVID-19 pandemic on the healthcare staff working in a fever clinic of a general hospital in Beijing using qualitative interviewing, and the Sources of Stress questionnaire (developed during the SARS outbreak in Hong Kong) and the Impact of Event Scale-Revised (IES-R) questionnaire.41 \u2018The rates of \u2026 clinically significant symptoms of anxiety, depression and PTSD were 34.3%, 31.2% and 24.5%, respectively\u2019. The factors that emerged as negatively correlated with participants reporting symptoms included availability of adequate PPE and well-being support, and lower exposure to moral dilemmas at work.Wanigasooriya et al report on an electronic survey of hospital healthcare workers in the West Midlands, UK using clinically validated questionnaires.45 They endeavoured to: quantify how the mental health of healthcare workers changed compared with before the pandemic; identify if healthcare workers on the front line, based in London or from ethnic minorities, had more severe symptoms compared with other staff; quantify the prevalence of severe psychiatric symptoms; and identify factors associated with those symptoms. They conducted a Qualtrics web-based survey open to all UK healthcare workers that presented the nine-item Patient Health Questionnaire (PHQ-9), seven-item anxiety scale (GAD-7), IES-R and Perceived Stress Scale to participants. Only healthcare workers \u2018\u2026 who had experienced a stressful or traumatic event related to COVID-19 were administered the IES-R. Also, the Connor-Davidson Resilience Scale (CD-RISC) was administered\u2019. \u2018Nearly a third of HCWs [healthcare workers] reported moderate to severe levels of anxiety and depression; and the number reporting very high symptoms was more than quadruple that pre-COVID-19\u2019 . Factors that influenced the results were reported to cluster within the themes of: demographics and roles; workplace readiness; risk management including PPE; experience of stressful events; protective experiences including sharing stress at work. The authors report that \u2018Front-line workers were significantly more likely to be more depressed, anxious, have high PTSD symptoms and be more stressed than non-front-line workers\u2026Working in London was associated with lower risk of depression \u2026 and anxiety \u2026 than outside London . Ethnic minority status (n\u00a0=\u00a0342) was significantly associated with greater risk of high PTSD symptoms (OR [odds ratio]\u00a0=\u00a01.52), but not high anxiety, stress or depression\u2019. \u2018Being able to share stress at work \u2026 [was] associated with significantly lower likelihood of being in the high anxiety, stress and depression groups \u2026\u2019.45Gilleen et al, looked at the impact of the pandemic on the well-being and mental health of healthcare workers in the UK.46 Data collection at time 1 was in November 2020  and at time 2 was in February 2021 . The validated questionaries were the GAD-7, PHQ-9, IES-R and Insomnia Severity Index. As the survey was open to new respondents at time 2, the authors report the initial findings as two cross-sectional studies. A high proportion of staff reported moderate-to-severe symptoms of depression, anxiety, PTSD and insomnia in the cross-sectional samples used at time 1 (26 to 30%) and time 2 (27 to 36%). Significantly more participants reported symptoms suggesting moderate-to-severe depression at time 2 but the other symptoms were not significantly different compared with time 1. These cross-sectional findings, which appeared to Jordan et al higher compared with findings from other studies of general UK and Irish populations, were \u2018broadly mirrored\u2019 in their longitudinal analyses comparing findings at times 1 and 2. But the authors also draw attention to another paper that found no significant differences between the prevalence rates for healthcarers and other populations.50Jordan et al report findings at two of four time points for which analyses are complete in an ongoing, online cross-sectional and longitudinal survey of the prevalence of mental health symptoms affecting health and social care staff in Northern Ireland.47 The authors report the \u2018prevalence of psychological distress among healthcare professionals was 78.3%\u00a0\u2026\u00a0[and prevalence] of insomnia was 50.2%\u2019. Interestingly, we note that higher rates of psychological distress were associated with not having a daily update on COVID-19 and feeling stigmatised or rejected because of working in a hospital.A hospital-based cross-sectional study was conducted in Ethiopia with 249 healthcare professionals by Yitayih et al.48 This study used the IES-R to measure distress, the State-Trait Anxiety Inventory (Form Y) to measure anxiety and the Maslach Burnout Inventory. The authors found that one-third of their participants had severe anxiety and distress. They draw attention to higher rates of distress in women and nurses. The authors conclude that detecting factors \u2018associated with worst psychological outcome may favour a tailored, preventive, organisational and psychological approach, representing a potential strength in counteracting the effects of future pandemics\u2019.Naldi et al, report a cross-sectional, survey of 797 healthcare workers in North-West Italy.49 They used the Depression Anxiety Stress Scales (DASS-21) and IES-R to measure symptoms. Their samples included doctors, nurses, administrative staff, pharmacists, cleaners, porters and technicians. The authors conclude that their study shows that \u2018the varied prevalence of psychological adversity among healthcare workers is independent of the burden of COVID-19 cases within \u2026\u2019 each of the countries studied. Interestingly, the authors say that Vietnam, which had the lowest volume of cases of COVID-19 per day per million population, also had \u2018a higher prevalence of PTSD related to COVID-19 among healthcare workers compared with India\u2019. Participants in Singapore \u2018reported the highest number of cases per day/1 million, but had a lower prevalence of depression and anxiety among its healthcare workers, when compared with the Malaysian cohort\u2019. Consistent with the opinions of other researchers, the authors of this paper comment on the potential beneficial effects from early psychological interventions for vulnerable groups of healthcare workers. These findings support our contention that other factors in addition to the risks introduced by COVID-19 were involved, as suggested by Fancourt et al and others.22Chew et al, report on a total of 1146 participants from India (384), Indonesia (250), Malaysia (175), Singapore (277) and Vietnam (60).51 This paper reports 3.2% of psychiatric providers were seropositive,51 whereas the proportion of staff in a tertiary medical centre were reported as 6.4%.52 Exposure at home predicted the presence of antibodies, but exposure at work did not. The authors recommend measures to prevent transmission from staff to patients in psychiatric facilities.One paper in the thematic series reports the first published serosurvey among psychiatric healthcare providers showing a lower seroprevalence compared with the results from other studies in the general population and other healthcare workers in Belgium.53 as a way of understanding and responding to the many psychosocial and mental health effects of the pandemic on staff.53 These interventions fall into the categories of: supporting the well-being of every member of staff (the Wellbeing Agenda); providing focused psychosocial interventions to meet the needs of staff who are struggling or have become distressed ; while remaining aware that a smaller number of staff may develop conditions that require specialist mental health assessment and, possibly, treatment . This fits with the findings in the papers we summarise and this approach to caring for staff is now included in guidance for the NHS in England from NHS England and NHS Improvement.54Another question raised by the thematic series is how should we develop reasoned approaches to caring for, and supporting healthcare staff? Murray et al suggest using a framework published before the pandemic22 But before the end of the first wave, cohesion had begun to falter, and the clapping stopped. Anecdotally, over the summer of 2020, UK staff began to take in the realities of what they had been through and became fatigued and/or disillusioned.22 Then came the second wave. Staff began to report anecdotally the difficulties in finding the energy and determination required to \u2018go again\u2019. Clearly, coping with the second and third waves has been a very different experience compared with the first.22The first wave of the COVID-19 pandemic was attended by huge public solidarity and mutual support in the UK. That support extended to staff in the NHS for whom the public clapped on Thursday evenings.55 The research team conducted qualitative interviews with 28 mental health professionals from varied professional backgrounds, career stages and across the UK who supported front-line healthcare staff. Many of them experienced professional growth. However, this came with the costs of additional responsibilities and increased workloads; many were professionally isolated and were affected vicariously by the experiences that healthcare workers talked about in interviews. Plainly, staff who support their colleagues also require care and support. A vital point to which this paper draws our attention is who cares for the carers?Many mental health providers have reached out to their colleagues and they, in turn, have come under strain. In this regard, the paper by Billings et al, is highly relevant.56 The authors found that many of the guidelines emphasised the importance of staff receiving psychological support and avoiding mental health disorders, whereas the healthcare workers placed greater emphasis on structural conditions at work, responsibilities outside the hospital and the support of the community . Similarly, Jordan et al draw attention to the consistency of their findings about the \u2018importance of organisational factors to staff well-being\u2019.46 Thus, Murray et al and other authors identify the importance of peer support.57 But the supporting interventions proposed in the guidelines reviewed by San Juan et al \u2018did not always respond to the lived experiences of staff, as some reported not being able to participate because of understaffing, exhaustion or clashing schedules\u2019.56 The authors concluded that \u2018Well-being guidelines should explore the needs of healthcare workers, and contextual characteristics affecting the implementation of recommendations\u2019.56 Although reporting a relatively focused study, this paper does show a way forward in meeting the needs of staff; the findings support the importance of community engagement and co-production in agreeing how to offer support.12At the beginning of the pandemic, many professional people were eager to express solidarity with their colleagues working at the front line by preparing guidance on how they should protect their psychosocial and mental health. But was this guidance helpful? San Juan et al, used qualitative methods to interview 33 front-line healthcare workers in the UK to look at the applicability of 14 sets of well-being guidelines in practice and provide recommendations for supporting front-line staff during the current and future pandemics.The papers in the themed series report studies conducted by a variety of research methods. Three papers report studies in which the authors used predominantly qualitative methods, 11 papers report the results of predominantly quantitative epidemiological methods, and the data in 2 papers derived from mixed methods. One paper offers a personal account, two are based on literature or narrative reviews, two offer findings from reviews of patients\u2019 case notes and one paper reports quantitative research on a clinic sample. Two of the papers used longitudinal approaches.46 Although providing useful pictures of symptoms experienced at, usually, a single time point, most of the quantitative studies in the series are based on cross-sectional analyses of data collected from convenience samples by self-report using screening tools. The limitations of these methods regarding the generalisability of the findings and making interpretations of causality are evident. Questionnaires and screening tools cannot be diagnostic and, especially when self-completed, tend to overestimate rates of disorders.1 Knudsen et al, make similar points when they make assertions about the importance of explicit sampling frames using known populations.18 Some, but relatively few, of the studies we present here have control populations or multiple sampling points. Also, many of the studies were conducted in the first months of the pandemic and have used short recruitment periods.All the papers report methods and sampling processes that preclude conclusions as to actual levels of diagnosed conditions or causality. In their paper, Jordan et al recognise some of the sampling challenges raised by studying restricted numbers of healthcare staff rather than representative samples of the whole workforce and raise the importance of longitudinal research.BJPsych Open published early papers as \u2018snapshots\u2019 or harbingers of what might be psychological and psychiatric concerns associated with the pandemic. A number of those papers identify what we call secondary stressors as important matters that have an impact on risk.11 This topic is raised in the paper by Murray et al who show its importance.22 Many of the studies reported in the papers in the series use additional questionnaires to explore factors that might bear on the experiences of staff working in healthcare services. The results from these questionnaires are very interesting and they serve as initial but incomplete presentations of pertinent stressors. We are tempted to draw from some of them impressions of the apparently positive adjustments of many healthcare staff based on these wider enquiries that contrast with the rates of symptoms that the screening tools indicate. This might fit with the possibility that participants were reporting distress and departures from well-being in self-reported surveys rather than symptoms of disorders. Necessarily, questionnaires control the discourse between participants and researchers, but participants may be tempted to fit their own experiences to the fixed questions asked. This suggests to us that mixed-methods research using both qualitative and quantitative approaches are required when researching novel experiences such as those the public and staff have faced in this pandemic.58The methodological limitations we cover also raise the importance of bias and noise in the investigations that further limit the conclusions that can be drawn from many of these studies. Biases, whether conscious or unconscious, tend to systematically shift sample means left or right in a distribution of scores whereas noise creates wider variability of scores within the distribution. Noise may occur within participants and between participants and we guess that \u2018level\u2019, \u2018pattern\u2019 and \u2018occasion\u2019 variations in scores that constitute noise are likely to occur in self-reported surveys.Another important methodological matter concerns the researchers\u2019 choices of instruments. It is important to be aware that choice of measures in studies is likely to reveal the researchers\u2019 assumptions about the definitions of distress and disorders and other features such as resilience. We opine that greater clarity of definitions, and greater rigour in explaining choice of measures are required.The Lancet, makes a similar point about distinguishing distress from disorder.59 It is also, for example, possible that more staff took part in some of the studies because they were concerned about their mental health and were, possibly, unwell.We conclude that most of the papers in our series may well describe a surge in distress, but we are unclear about levels of both persistent distress and rates of disorders and the methods used do not allow us to distinguish distress from disorders. Smith, Editor-in-Chief of 18 point out that chronic stressors and economic recession may be factors that have a long-term impact on the mental health of the public. In this context, the papers in the thematic series present data from, mainly, the first wave of the pandemic. We recognise that the early impact may be rather different compared with the longer-term impact, and we know from research into other contrasting events, such as 9/11, just how long some people's serious mental health disorders may go on and how delayed may be their presentations.2 Therefore, it is important to monitor research conducted during subsequent waves and for many years afterwards. We are aware, for example, that research on paramedics and emergency medical technicians involved on 9/11 into the long-term impact on their physical and mental health bear out this matter.60 The study by Smith & Burkle was conducted in 2016 \u2013 15 years after 9/11; it documents the seriousness of the enormous long-term mental and physical health effects on ambulance staff of their participation in caring for people involved in that event.60 In the short-term, we should be clear that distress is not equivalent to mental health disorders. In the longer-term, studies of distress, mental health and physical health disorders are needed to examine whether there is deterioration of public mental and physical health consequent on the pandemic.Knudsen et alBJPsych Open recognises the, arguably, inevitable limitations of many studies conducted early in the pandemic, it also recognised at the time that there was a desire for initial knowledge and, consequently, published a number of papers to offer readers access to initial findings. Our longer-term goal is to include longitudinal studies with comparators to better define the development of symptoms, disorders and causality as time elapses and opportunities arise in and after the pandemic. Therefore, we emphasise the need for longitudinal and clinical research studies over a much greater time that incorporate comparisons with similar groups pre-pandemic or that use matched controls.Nonetheless, although to BJPsych Open that offer a picture of the ways in which populations and healthcare workers have been affected in the opening months of a much longer-term emergency. They claim high prevalence of symptoms of anxiety, depression and post-traumatic stress. Jordan et al remark that the prevalence figures from their work with health and social care staff are higher than those from studies of the public. But they also draw attention to a paper from C\u00e9nat et al that found no significant differences between the prevalence rates for healthcarers and other populations.50 The methods used in many studies do not allow us to form any definite conclusions. We, as Knudsen et al and Appleby, express caution.19 But pragmatically, we are learning a huge amount about the value of, and best methods for supporting the public and our colleagues. There can be no doubt of the importance of relationships in that task as a number of the papers point out. One of the papers identifies that post-traumatic growth is another possibility.5A huge amount of research has been done very rapidly in the pandemic. We are eager to bring together this thematic issue because it illustrates the papers that have been presented As the fourth wave continues in the UK, we remember that other countries may be in different phases. Even if the COVID-19 pandemic can be brought under control through vaccination across the world, and that will take a long time yet, we must face the economic effects that create chronic stressors that may lead to mental health problems. This is the time to pursue more research and to employ methods that are appropriate to addressing long-term distress, mental health symptoms and disorders, protective and risk factors, and the development and treatment of long-term neuropsychiatric sequelae of COVID-19. The papers included here have another important function; they inform us about refining research methods with a view to producing more definitive learning."}
+{"text": "Throughout the coronavirus disease 2019 (COVID-19) pandemic, health and social care workers have faced unprecedented professional demands, all of which are likely to have placed considerable strain on their psychological well-being.To measure the national prevalence of mental health symptoms within healthcare staff, and identify individual and organisational predictors of well-being.n = 3834) and time 2  results are presented here. At time 2, 84% of respondents had received at least one dose of a COVID-19 vaccine. The survey included four validated psychological well-being questionnaires , as well as demographic and organisational measures.The COVID-19 Staff Wellbeing Survey is a longitudinal online survey of psychological well-being among health and social care staff in Northern Ireland. The survey included four time points separated by 3-month intervals; time 1 , anxiety (26\u201327%), post-traumatic stress (30\u201332%) and insomnia (27\u201328%); overall, significance tests and effect size data suggested psychological well-being was generally stable between November 2020 and February 2021 for health and social care staff. Multiple linear regression models indicated that perceptions of less effective communication within their organisation predicted greater levels of anxiety, depression, post-traumatic stress and insomnia.This study highlights the need to offer psychological support to all health and social care staff, and to communicate with staff regularly, frequently and clearly regarding COVID-19 to help protect staff psychological well-being. During the same time period, lower caseness estimates for depression (15%) and anxiety (12%), but higher rates of post-traumatic stress (35%) were reported among medical and nursing staff in China. Exposure to unique stressors and wider organisational strain, including \u2018moral injury\u2019 a source of psychological distress related to clinical pressures and decision-making that violates a staff member's moral or ethical code3 may partially account for these enhanced mental health difficulties in healthcare staff.2The coronavirus disease 2019 (COVID-19) pandemic represents one of the most significant global threats to societal, physical and mental health in over a generation. Evidence from representative community studies indicate that the general population in the UK have experienced clinical levels of a range of psychological symptoms, including anxiety (22%), depression (22%) and post-traumatic stress (17%).5 Healthcare staff in front-line positions involving direct contact with patients with COVID-19 are also at higher risk of psychopathology.6 Moreover, such organisational variables are likely to interact with personal factors such as age; professional experience; personal coping styles; family exposure to COVID; and pre-existing psychological difficulties that influence vulnerability to distress.7Data from previous outbreaks and the current COVID-19 crisis suggest that both organisational and individual factors can mitigate the psychological impact of the pandemic on health workers. Mental health burden can be offset by workplace measures such as clear communication; supportive team networks; access to adequate personal protection equipment (PPE); provision of relevant training for job role; and access to appropriate psychological support.8 as opposed to representative samples of the entire healthcare workforce, including neglected subgroups such as domestic and support services. There is also a widely acknowledged need to move away from stand-alone cross-sectional studies and towards longitudinal methodologies examining the mechanism and course of mental health symptoms in staff over time.2 Moreover, risk factors and protective buffers within healthcare staff and their parent organisations need to be identified and tracked in order to ensure the development of timely, nuanced staff well-being support strategies.Despite the rapidly evolving literature base on COVID-19-related mental health difficulties in healthcare staff, there remain a number of gaps in empirical understanding. Several prominent studies have focused on a restricted number of healthcare professions (for example medics and nurses)A key aim of the present exploratory study was to examine the impact of organisational, demographic and profession-specific factors on mental health. Online survey methodology was used to measure the national prevalence of mental health symptoms in health and social care staff as well as other relevant individual and organisational factors. It provides findings from the first two time points (3 months apart) of a larger longitudinal study examining changes in staff well-being during and after the second wave of the COVID-19 pandemic.The COVID-19 Staff Wellbeing Survey was open to all health and social care staff working in Northern Ireland. In Northern Ireland, both health and social care are provided by one organisation, in contrast to England where healthcare services are provided by the National Health Service and social care by local councils. The design incorporated both cross-sectional and longitudinal elements and spans four time points: time 1 (November 2020), time 2 (February 2021), time 3 (May 2021) and time 4 (August 2021). The time point spacing was designed to cover anticipated phases of the pandemic , minimise survey fatigue effects and allow for service development in response to findings between time points.9 were employed in health and social care roles in Northern Ireland, and were therefore eligible to take part. Of these staff, the cross-sectional sample sizes were 3834 at time 1 (response rate 4.9%) and 2898 at time 2 (response rate 3.7%). At time 1, a total of 5385 staff started to complete the survey with 71% of these completing it \u2013 further examination highlighted that respondents gradually dropped out throughout the survey and no specific question was particularly associated with drop-out. Staff were given the option of leaving their email address at each time point to enable their responses to be linked over time; a longitudinal data-set was created comprising the 632 staff who submitted their email address at times 1 and 2.Two time points have been completed thus far with data collection taking place during 9\u201322 November 2020 (time 1) and 8\u201328 February 2021 (time 2). Staff were recruited via a broad range of methods including broadcast emails to all staff; emails to staff who left an email address at time 1; posts on staff twitter and Facebook; laminated posters in staff areas; and screensaver messaging. At the time of data collection, approximately 78\u00a0000 staffThe COVID-19 Staff Wellbeing Survey collected a broad range of data including demographics; caring responsibilities; job satisfaction; psychological well-being; redeployment experiences; COVID-19 risk factors and exposure; environmental needs; communication; accessed mental healthcare services; and future psychological needs. The focus of this article is on the four psychological well-being outcome measures.10 depression ,11 post-traumatic stress 12 and insomnia .13 Established cut-off scores were used to designate symptoms as moderate-severe on these measures: \u226510 for GAD-7 and PHQ-9; \u226526 for IES-R; and \u226515 for ISI.14 The participants were instructed to complete the IES-R with \u2018respect to the COVID-19 outbreak\u2019.The constructs measured included anxiety ,https://doi.org/10.1192/bjo.2021.988.The following variables were used as predictor variables of psychological well-being in the regression analyses: occupation, gender, age, COVID-19 exposure; if they managed patients with COVID-19; if they have one or more risk factors for COVID-19 (such as diabetes); perceived effectiveness of communication by their organisation on COVID-19-related matters, if they were asked to consider a redeployment opportunity; and if vaccinated (time 2 only). All binary predictors were coded as follows: 0,\u00a0no; 1,\u00a0yes. Further details on the psychological well-being outcome and predictor variables are included in Supplementary Table 1 available at Respondents voluntarily completed the survey online via the Survey Mechanics platform. At time 2 they were instructed that they could take part even if they had not participated at time 1. The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. All procedures involving human patients were approved by West of Scotland Research Ethics Service (REC reference 20/WS/0122). Participants indicated their consent to participate by clicking to start the questionnaire after reading the online information sheet. Participants were free to withdraw from the study at any stage while completing the questionnaire up until they clicked \u2018submit\u2019 at the end of the questionnaire. Both the information sheet and the final page of the questionnaire provided details of individuals they could contact regarding psychological well-being support.2-test for categorical variables and independent t-tests for continuous variables. We used t-tests on the cross-sectional (independent t-tests) and longitudinal (paired t-tests) samples to examine change over time on total scores of the psychological well-being measures. Chi-square  and McNemar  tests provided an assessment of change over time in the proportion of health and social care staff reporting moderate-to-severe depression, anxiety, post-traumatic stress and insomnia symptoms.All analyses were conducted using IBM SPSS Statistics version 26 for Windows. The demographic profiles of the time 1 and 2 cross-sectional samples were compared using the \u03c7The purpose of the longitudinal analysis was to check if changes in the cross-sectional sample were replicable or likely because of differences in the composition of the samples at the two time points. Multiple linear regression models with simultaneous entry were then used to examine predictors of psychological well-being for the time 1 and 2 cross-sectional samples.All survey questions were mandatory (except email address); hence there were no missing values on any of the variables reported here, except for age where a small number of impossible values were recorded . In the regression models that included age as a covariate, listwise deletion was used.n\u00a0=\u00a03834) and 2 (n\u00a0=\u00a02898) for the cross-sectional samples are presented in 15 that shows that women comprise four-fifths (79%) of the workforce. The age profile of the time 1 sample and the health and social care staff profile is reasonably similar  reporting being educated to level four (such as university degree) or above.Participant characteristics at time 1 . However, with the longitudinal sample the over-representation of administrative and clerical, and professional and technical staff was greater, as was the underrepresentation of nursing and midwifery staff.15 (figures exclude care home and senior executives) the achieved sample has good representation from most sectors. Groups of staff with more desk-based roles such as administrative and clerical (time 1 28% v. population data 19%) and professional and technical (time 1 20% v. population data 15%) who would have had greater access to computers, unsurprisingly tended to be over-represented in the survey whereas those with greater patient contact (such as nursing and midwifery (time 1 24% v. population data 33%) were underrepresented.A large proportion of the participants worked in administrative and clerical (28%), nursing and midwifery (24%), and professional and technical (20\u201321%) roles. Compared with occupational distribution data for health and social care staffSupport services/user experience were the most underrepresented in the present sample; this sector typically comprises approximately 10% of the health and social care workforce, five times the proportion achieved in time 1 of the COVID-19 Wellbeing Survey.A high proportion of staff reported moderate-to-severe symptoms of depression, anxiety, PTSD, and insomnia in the time 1 (26\u201330%) and 2 (27\u201336%) cross-sectional samples .Fig. 1PP\u00a0<\u00a00.001, d\u00a0=\u00a00.12); no significant difference was evident for anxiety , post-traumatic stress  or insomnia .Comparisons of the cross-sectional samples (Supplementary Tables 3 and 4) revealed a significantly higher proportion of respondents reporting moderate-to-severe depression at time 2 than time 1 \u00a0=\u00a0\u22124.84, P\u00a0<\u00a00.001, d\u00a0=\u00a00.12), post-traumatic stress (t(6127.42)\u00a0=\u00a0\u22122.42, P\u00a0=\u00a00.016, d\u00a0=\u00a00.06) and insomnia (t(6730)\u00a0=\u00a0\u22122.06, P\u00a0=\u00a00.039, d\u00a0=\u00a00.05), but not for anxiety (t(6730)\u00a0=\u00a0\u22121.01, P\u00a0=\u00a00.312, d\u00a0=\u00a00.02). All comparisons between the cross-sectional samples yielded small effect sizes.The time 1 and 2 samples were also compared using the total scores on the four psychological well-being measures; significantly poorer well-being was evident in the time 2 sample compared with the time 1 sample for depression (P\u00a0=\u00a00.071), anxiety (P\u00a0=\u00a00.694), post-traumatic stress (P\u00a0=\u00a00.863) or insomnia (P\u00a0=\u00a00.395) in the subsample for whom we had longitudinal data. Paired t-tests showed no significant change over time for depression (t(631)\u00a0=\u00a0\u22121.94, P\u00a0<\u00a00.053, d\u00a0=\u00a00.08), post-traumatic stress (t(631)\u00a0=\u00a0\u22120.45, P\u00a0=\u00a00.656, d\u00a0=\u00a00.02) or anxiety (t(631)\u00a0=\u00a00.01, P\u00a0=\u00a00.992, d\u00a0=\u00a00.00); a small but significant increase in insomnia symptoms was observed (t(631)\u00a0=\u00a0\u22122.34, P\u00a0=\u00a00.020, d\u00a0=\u00a00.09).Comparable analyses were performed using the longitudinal sample (see Supplementary Tables 3 and 4). In keeping with the cross-sectional results, descriptive data for the longitudinal samples follow the trend of poorer well-being at time 2 compared with time 1. However, statistical analyses using McNemar tests showed no significant difference over time in the proportion reporting moderate-to-severe symptoms for depression  and 2 . Both se\u03b2\u00a0=\u00a0\u22120.19 to \u22120.25).At both time points, a significant relationship was evident between at least two of the four psychological well-being measures and the organisational/risk factor variables. Specifically, poorer psychological well-being was associated with managing patients with COVID-19, having had higher exposure to COVID-19, having at least one COVID-19 risk factor, perceiving the communication from their organisation to have low effectiveness and being asked to consider a redeployment opportunity. Across both time and psychological well-being measures, the perceived effectiveness of communication by their organisation on COVID-19-related matters was the strongest predictor of well-being , we found high rates of depression, anxiety, post-traumatic stress and insomnia. It is the first study on healthcare staff to report longitudinal findings before and after staff have received their first vaccination. Across the two time points many staff reported moderate-to-severe levels of depression on the PHQ-9 , anxiety on the GAD-7 , PTSD on the IES-R  and of insomnia on the ISI . The results of cross-sectional analysis were broadly mirrored in the longitudinal analyses in that the psychological distress levels remained consistently high across the time points; where significant differences did occur the effect sizes were very small.16 Shevlin et al1 report rates of moderate-to-severe depression on the PHQ-9 of 22%, that of anxiety on the GAD-7 of 22% and that of post-traumatic stress on the International Trauma Questionnaire of 17%. Our results are broadly in keeping with the higher end of estimates of caseness among healthcare workers elsewhere during the COVID-19 pandemic.The rates reported here appear higher than those in the general UK and Irish populations during the first year of the pandemic.6 However, we do note a recent review of populations affected by COVID17 that found no significant differences between healthcare workers and other populations affected by COVID-19 on measures of depression, anxiety and PTSD but twice the levels of insomnia \u2013 all groups in their analysis experienced much higher rates than would be expected.Worldwide studies have demonstrated very significant levels of anxiety, depression, insomnia and post-traumatic stress in healthcare workers with estimates of caseness ranging from 15 to 27% for depression, 12 to 23% for general anxiety and 30 to 35% for post-traumatic stress symptoms.20 we found a range of individual and organisational variables have a role in predicting distress at both time points. Importantly, a strength of our sample was that it included all roles and jobs within the health and social care system in Northern Ireland. An important finding was that at time 1, administrative and clerical staff, and support services staff  had greater anxiety, depression and post-traumatic stress than nursing and midwifery staff. At both time points, we found a significant association between at least two of the four psychological well-being measures and the organisational/risk factor variables. Across both time and psychological well-being measures, the perceived effectiveness of communication by their organisation on COVID-19-related matters was the strongest predictor of well-being.In terms of predictors of distress, in keeping with previous literature,Vaccination uptake at time 2 did not predict well-being. It should be noted that the predictive models explained 10\u201312% of the variation in the four psychological well-being measures, meaning other factors not tapped by the models clearly contribute to staff well-being as well.21 Examples of interventions include psychological assistance hotlines, online courses and group activities to help with stress.22 Interventions may also include preventative approaches and the provision of timely and accessible individual mental health treatments in cases of emerging mental health problems.23The high rates of distress are in keeping with the need to provide interventions and prevention strategies to all types of healthcare workers both during this pandemic and as health systems are recovering from it. Despite the majority of our sample receiving their first vaccination at time 2 this did not appear to improve staff mental health. It appears organisations cannot rely on a vaccine \u2018bounce\u2019 to improve the well-being and mental health of their staff. While the evidence regarding effective staff support interventions is relatively sparse there is a need for intervention strategies to be developed at an individual, team and organisational level.This study highlights that the provision of staff support interventions should not just be targeted at staff that are exposed to COVID-19 or that are working with patients with COVID-19. The results demonstrated that administration staff (secretaries and receptionists) as well as staff involved in support services  were at higher risk of distress than other staff groups. An effective health service needs a wide variety of jobs and roles to function effectively. It is imperative support interventions are available and accessible to all.25 This may very well be more important in a pandemic. By its very nature the situation is often entirely new to staff and guidance can change on a daily basis. Several professional bodies in the UK have highlighted the importance of a communication strategy to staff well-being and the importance of communicating with staff regularly, frequently and in simple clear ways.26 Muller et al,21 in a recent review, do note the frequent mismatch in studies of staff support interventions of the likely organisational sources of distress  and the frequent focus on relieving distress at an individual level.The findings are entirely consistent with a body of research highlighting the importance of organisational factors to staff well-being.16 Although there have been few psychiatric epidemiological studies in Northern Ireland to compare our rates with, the one exception is rates of PTSD. The Northern Ireland Study of Health and Stress, part of the World Mental Health Survey Initiative previously reported levels of PTSD in Northern Ireland of 5%.27 Our current rates of PTSD, as measured by the IES-R are considerably higher.The starting point of this study was during the second wave of the pandemic (November 2020) and the second time point was February 2021. An obvious limitation is the lack of pre-pandemic baseline of staff mental health. However, as stated earlier we can compare rates with a number of studies of the general population in the UK and Ireland during the pandemic.It is strength of our study that we included all staff groups. However, there was low uptake from some occupations (such as support services) meaning that the rates cannot be used as precise \u2018prevalence rates\u2019 for the whole of the health and social care sector. Rather they provide a general indication of the level of need. In staff surveys in Northern Ireland that were run pre-COVID-19, response rates have tended to be lowest in this sector, as they can be particularly hard to reach (i.e. no work email addresses). Engaging with this group during a pandemic has become even more challenging because of infection control rules . Given that the group who were most underrepresented tended to have poorer mental health, the overall prevalence figure may be an underestimation of levels of distress among staff.6A further limitation is that our indicators of mental health are based on survey self-report data rather than diagnosis based on clinical interviews. We have, however, used instruments with good psychometric properties and our methodology is in keeping with all other studies of staff mental health during the pandemic that we are aware of. It should also be acknowledged that there is a lack of consensus regarding established clinical cut-offs for use with the IES-R; to allow for international comparisons we adopted that used by Lai et al.In conclusion, this study is one of the first longitudinal studies of health and social care staff mental health and well-being during the COVID-19 pandemic to be published. It strengthens the argument about the need to provide a comprehensive system of staff supports to all health and social care staff during and post this pandemic. This would appear essential if health services begin to recover function following this global pandemic."}
+{"text": "Staphylococcus aureus (MRSA) is an important pathogen for human infection. Hospital-acquired (HA) and community-acquired (CA) MRSA infections are serious clinical problems worldwide. In this study, we selected typical HA-MRSA strain and CA-MRSA isolates from our previous research and compared their phenotypic and pathogenic abilities both in vitro and in vivo.Methicillin-resistant mecIVa (YNSA7) and ST59-t437-SCCmecVb (YNSA53) belonged to two prevalent subclones of CA-MRSA, while ST239-t030-SCCmecIII (YNSA163) was an HA-MRSA epidemic clone in Southwest China. ST59-t437 strains demonstrated faster growth ability, higher survival rate resistance to human blood, and more toxin secretion levels and cytotoxicity than ST239-t030. The virulence and regulatory genes of hld, psm-\u03b1, RNAIII, agrA, and crtN were highly expressed on CA-MRSA isolates, especially the ST59-t437-SCCmecIVa subclone. However, the ST239-t030 strain had the strongest adhesion and biofilm ability among these MRSA bacteria. Animal experiments revealed the most serious lethal effect on BALB/c mice caused by the YNSA7 strain infection. The survival rates of BALB/c mice infected with the three MRSA strains were 16.7, 50.0 and 100.0% for YNSA7, YNSA53 and YNSA163, respectively. Histopathological analyses of infected animals indicated that the lungs were the most seriously damaged organs, especially for ST59-t437 MRSA. Severe inflammatory reactions, tissue destruction, and massive exudation of inflammatory mediators and cells could be identified in ST59-t437 strain-infected animals.ST59-t437-SCCIn general, ST59-t437 strains showed higher pathogenic ability than the ST239-t030 isolate, while ST239-t030 MRSA revealed the features prevalent in hospital settings, specifically for adhesion and biofilm ability. Staphylococcus aureus (MRSA) has increased globally [Recently, the incidence of methicillin-resistant globally ; MRSA haglobally , 3. Thisglobally . BecauseS. aureus are pulsed-field gel electrophoresis (PFGE) [spa typing and staphylococcal cassette chromosome mec (SCCmec) typing [S. aureus. MLST (ST type) is suitable for analysing the evolutionary relationship and clonal complexes of strains. Other methods also show high discriminatory abilities and usage. In addition, the complete genome sequencing method has become the gold standard for analysing the genomic diversity of bacteria [The major molecular typing methods for s (PFGE) , multilos (PFGE) , spa typ) typing . PFGE isbacteria . At presbacteria . Among tbacteria .Our previous study revealed that ST59-t437 and ST239-t030 were the major genotype profiles of MRSA for human infections in Southwest China . The ST2MRSA has many pathogenic mechanisms , 12. HowmecIVa genotype, and YNSA53 was ST59-t437-SCCmecVb, as shown in Table\u00a0mecIII type, isolated from an old hospitalized case from the Department of Respiratory Medicine, and the strain sample type was sputum. All these cases were cured and discharged after clinical treatments.The two CA-MRSA strains used in this study were isolated from young patients under 5 years old from the Department of Paediatrics, and both cases had pneumonia. The sample types of the isolated strains were nasopharyngeal aspirate and sputum. The YNSA7 isolate showed the ST59-t437-SCCBoth ST59-t437 strains in this study showed identical antibiotic resistance profiles, and all were sensitive to gentamicin (GEN), rifampicin (RIF) and vancomycin (VAN). However, the ST239-t030 isolate revealed different resistance results from ST59-t437 MRSA, as shown in Table\u00a0Comparison of the growth curves of the ST59-t437 and ST239-t030 strains indicated that the YNSA7 and YNSA53 isolates grew faster than the YNSA163 strain before 24\u2009h in vitro, especially during the log phase. Statistical significance was found at 4, 6, 8, 10, 12, 14, 16 and 18\u2009h between the two ST59-t437 strains and the ST239-t030 isolate, as shown in Fig.\u00a0P\u00a0=\u20090.000), while no significant difference was found between the two ST59-t437 isolates by using the LSD method of the post hoc test in this study , while YNSA7 and YNSA53 were significantly different from YNSA163 (P\u2009=\u20090.000).The staphyloxanthin assay revealed that the YNSA7 and YNSA53 strains produced more staphyloxanthin than YNSA163 , as shown in Fig. After bacteria were incubated with healthy whole blood from humans, 15.92\u2009\u00b1\u20093.51% of the YNSA7 strain survived after the experiment, and the survival rate for YNSA53 after incubation was 7.79\u2009\u00b1\u20091.05% and 7.69\u2009\u00b1\u20092.57% for the YNSA163 strain. A higher survival rate of YNSA7 was identified compared with YNSA53 and YNSA163 . The survival rates were 29.29\u2009\u00b1\u20092.92%, 37.1\u2009\u00b1\u20091.19% and 33.29\u2009\u00b1\u20096.15% for YNSA7, YNSA53 and YNSA163 after H2O2 killing, respectively . YNSA7 showed the worst cell viability among the three strains, followed by YNSA53 and YNA163 , followed by YNSA 53, and YNSA7 showed the lowest biofilm formation ability , and YNSA53 and YNSA163 also showed significant differences (P\u2009=\u20090.000).The light microscopic examination results for the biofilms for three MRSA strains were shown in Fig.\u00a0ity Fig. . The staThe animal experiments revealed the most serious lethal effect on BALB/c mice caused by YNSA7 strain infection. At day 2 after bacterial injection, just one animal died; however, at day 3 of infection, three mice died. Finally, the survival rate of BALB/c mice infected with the YNSA7 isolate was 16.7% Fig.\u00a0. Only onHistopathological analyses of the tissues and organs of infected BALB/c mice indicated that the lungs were the most seriously damaged organs. Extensive tissue necrosis, pyknosis or fragmentation of nuclei, and widespread eosinophilic homogenate (black arrow) could be found by infection with the YNSA7 strain Fig. . The strSeveral factors contribute to the success of MRSA as a pathogen and provoke the host\u2019s innate and adaptive immune responses during infection . Some seCA-MRSA usually causes soft tissue and skin infections, abscesses and folliculitis, especially in young patients . For sommecIVa clone showed higher pathogenicity than the ST59-t437-SCCmecVb clone, and more toxin levels and serious lethality of animals could be found for the ST59-t437-SCCmecIVa clone. The gene annotations of previous research showed that both ST59-t437 isolates in this study had PVL genes. However, the transcriptome sequencing of three strains for differential gene expression (our unpublished data) revealed no significant difference in PVL gene expression between the two ST59-t437 MRSA strains in this study. Therefore, we considered that other factors might contribute to the enhanced pathogenic ability of the ST59-t437-SCCmecIVa strain.CA-MRSA clones have evolved in different geographic regions. ST8 is currently the most prevalent one in the United States , 21. HowmecIVa might illustrate the higher pathogenicity of the strain. Regulations of gene expression play an important role in the pathogenesis of MRSA strains. The accessory gene regulator (agr) is a quorum sensing system that plays a critical role in the regulation of MRSA virulence. A previous study showed the decreased virulence found in agr mutant strains and certain types of agr led to specific clinical syndromes [S. aureus: the psm-\u03b1 operon, encoding PSM\u03b11\u20134; the psm-\u03b2 operon, encoding PSM\u03b21\u20132; and hld, encoding \u03b4-toxin. Hld is also part of the coding sequence of RNA III, the master regulatory RNA in staphylococci [mecIVa clone compared with the ST59-t437-SCCmecVb clone and ST239-t030-SCCmecIII clone in this study.The upregulated expression of hld, psm-\u03b1, RNAIII, and agrA genes on ST59-t437-SCCyndromes . Phenol yndromes . For exayndromes . PSMs arylococci . All theIn addition, pathological examination results showed that the strains mainly attacked the lungs of the host, especially the ST59-t437 strains, which caused severe inflammatory reactions, tissue destruction, and massive exudation of inflammatory mediators and cells. All these changes resulted in ventilatory and ventilation dysfunction of the host, leading to the death of the animal.In this study, we selected and compared the phenotypic and pathogenic abilities among prevalent clones of MRSA in Southwest China, including ST59-t437 and ST239-t030. ST59-t437 strains showed higher pathogenic ability than the ST239-t030 isolate both in vitro and in vivo, while ST239-t030 MRSA revealed the features prevalent in hospital settings, specifically for adhesion and biofilm ability. These results might partially indicate the reasons for the prevalence of different clones in environments and provoke different clinical infections.http://www.genomicepidemiology.org).The MRSA strains used in this study were shown in Table S. aureus ATCC 29213 was used as a quality control [Thirteen antibiotics were determined through the broth microdilution method using customized microtiter plates  according to the manufacturers\u2019 instructions. The antibiotics tested were penicillin (PCN), oxacillin (OXA), gentamicin (GEN), ciprofloxacin (CIP), levofloxacin (LEV), moxifloxacin (MXF), erythromycin (EM), clindamycin (CM), linezolid (LZD), vancomycin (VAN), tetracycline (TET), rifampicin (RIF) and trimethoprim/sulfamethoxazole (SXT). The tests were performed and interpreted in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines M100, 2018), and 0, 2018, 5\u2009CFU/mL bacterial suspension). The isolates were kept at 37\u2009\u00b0C with shaking, and the OD values were measured at 600\u2009nm every 2\u2009h [To compare the growth curve between ST239-t030 and ST59-t437 in this study, 20\u2009mL of TSB was inoculated with 100\u2009\u03bcL of MRSA strains cultured for 16 h  and kept at 37\u2009\u00b0C with shaking for 24\u2009h. The bacteria were centrifuged at 12,000\u00d7g for 5\u2009min, and the supernatants were removed. The pellets were resuspended by methanol extraction under shaken conditions at 37\u2009\u00b0C overnight. The extracted staphyloxanthin was detected at OD 462\u2009nm [Twenty mL of TSB was inoculated with 100\u2009\u03bcL of a 16-h culture of MRSA strains  at a ratio of 1:4 and incubated at 37\u2009\u00b0C for 3\u2009h with shaking. The surviving isolates were calculated by the plate counting method as described previously [MRSA strains were grown on TSA medium, and 10eviously , 36.8\u2009CFU of isolates were adjusted by using saline. The bacterial suspensions were treated with 0.2% H2O2 at 37\u2009\u00b0C for 3\u2009h. Then, the plate counting method was used to analyse the viable surviving bacterial cells [MRSA strains were grown on TSA medium, and 105 cells/mL) cultured with DMEM (Thermo Scientific) with glutamine supplemented with 10% foetal bovine serum (Thermo Scientific) at 37\u2009\u00b0C containing 5% CO2 for 24\u2009h. A concentration of 105\u2009CFU/mL strains was used, and 100-\u03bcL bacterial suspensions were added to Hep2 cells and then cocultured for 48\u2009h at 37\u2009\u00b0C. After incubation, the plates were washed with PBS buffer (pH\u20097.4) three times [Monolayer Hep2 cells were prepared by seeding 96-well plates with 100\u2009\u03bcL of cell suspension . Expression analysis was performed using a CFX-96 real-time PCR instrument  for sarA, aur, nuc, agrA, fnbA, RNAIII, hld, psm-\u03b1 and crtN, referring to virulence and biofilm formation . Real-tiThe biofilm assay was performed according to a previous study . BrieflyFor light microscopic examination, the washed plates were stained with 0.4% crystal violet, washed with PBS buffer to remove the excess stain, and then air-dried. Finally, the plates were examined under a light microscope at a magnification of 200\u00d7 .MRSA strains were cultured for 16 h, diluted with TSB 100 times, and then seeded into 12-well plates containing sterile coverslips. The strains were cultured for 24\u2009h at 37\u2009\u00b0C, and then, the plates were washed three times with sterile PBS buffer (pH\u20097.4). The coverslips were fixed with 2.5% glutaraldehyde solution for 30\u2009min at room temperature and washed with PBS buffer three times. Finally, the coverslips were dehydrated by an ethanol gradient , dried and ion plated, and then observed by SEM  .8\u2009CFU of three MRSA strains. The control group of mice received PBS with the same volume of infected groups. The animals were observed daily for 1 week, and at 7 days after infection, the BALB/c mice were euthanized by cervical dislocation. Histopathological analyses were performed on the hearts, livers, spleens, lungs and kidneys of animals, and the tissues were fixed with 4% paraformaldehyde, stained and examined by using the HE method.BALB/c mice were used for in vivo experiments and obtained commercially from the Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College. The animals were divided into four groups, containing three MRSA isolate-infected groups and one control group. Each group had six animals. Mice were infected via intravenous injection with 2\u2009\u00d7\u200910P value <\u20090.05 was considered to indicate statistical significance.Statistical analyses were performed using SPSS . The Kolmogorov-Smirnov test, t-test, ANOVA, and Kruskal-Wallis H test was used as appropriate. A"}
+{"text": "Haliotis discus hannai. Phylogenetic and exon\u2013intron arrangement analyses of bilaterian NPF and the chordate ortholog NPY with their receptor sequences revealed a likely common ancestor, and Hdh-NPFR was similar to the NPYR2 subtype among the NPYR1, NPYR2, and NPYR5 subtypes. Among four Hdh-NPFR-related receptors, Hdh-NPFR specifically responded to Hdh-NPF peptide, supported by the dose\u2013response luciferase reporter curve, intracellular Ca2+ mobilization, and phosphorylation of ERK1/2 and its inhibition with a protein kinase C inhibitor. Peptide fragmentations and shuffling of Hdh-NPF with human NPY could not activate the cellular response of Hdh-NPFR. Three-dimensional in silico modeling suggested that interaction of Hdh-NPF C-terminal amino acids with the extracellular loops of Hdh-NPFR is critical for Hdh-NPFR activation. In vivo injection of Hdh-NPF peptide increased food consumption, and knockdown of Hdh-NPF expression decreased food consumption in Pacific abalone. These findings provide evidence for co-evolution of the NPF/Y ligand-receptor system, enabling further research on mollusk orexigenic neuropeptides.Neuropeptides function through G protein-coupled receptors (GPCRs) with high specificity, implying a significant degree of neuropeptide-GPCR coevolution. However, potential neuropeptide signaling systems in non-chordates are relatively elusive. We determined the specificity of the neuropeptide F (Hdh-NPF) signaling system with a cognate receptor (Hdh-NPFR) in the Pacific abalone,  The evolutionary origins of NPs have been traced to the common ancestor of protostomes and deuterostomes, in which two rounds of genome duplication in the vertebrate lineage gave rise to an expanded repertoire of NP signaling systems with cognate receptors4. For example, identification of the [LV]Wamide  cluster in the eumetazoan species has shed light on the complex mechanism of duplication and gene loss for the origin of these NP families, such as the bilaterian adipokinetic hormone (AKH) and gonadotropin-releasing hormone (GnRH)/corazonin (CRZ) families5. Given the conserved role of GnRH in reproductive endocrine systems, lineage-specific loss of CRZ signaling in vertebrates and duplication of the GnRH signaling system with AKH in invertebrates may account for the global phylogenetic distribution of AKH/CRZ/GnRH-type receptor pathways6.Neuropeptides (NPs) synthesized and secreted by neurons are essential regulators in diverse animal phyla. NPs exert their effects locally as hormones, neurotransmitters, and neuromodulators for various physiological functions, including growth, metabolism, and reproduction7, belongs to the NPY family of biologically active peptides, together with two other members: peptide YY (PYY) and pancreatic polypeptide (PP)8. In vertebrates, NPY plays an essential role in diverse physiological functions, including food intake, energy homeostasis, anxiety, and stress responses, through interaction with NPY receptors (NPYRs)11, which belong to class A G-protein coupled receptors (GPCRs), activated by the closely related peptides NPY, PYY, and PP12. Activation of vertebrate NPYRs by NPY primarily results in decreased cyclic adenosine monophosphate (cAMP) accumulation through Gi protein, leading to the inhibition of adenylyl cyclase in mammalian cells12. Furthermore, human NPY-activated NPYRs induced increases in intracellular Ca2+ levels possibly through Gq protein14. Recognition experiments of mammalian NPY by its receptors using chemically modified NPYs, receptor mutagenesis, and receptor chimeras demonstrated that NPYRs require both ends of NPY for binding, and intact Tyr-32, Arg-35, and Tyr-36-NH2 residues are critical for the fully active form16. Small-molecule compounds that regulate NPY signaling systems have pharmaco-therapeutic implications, with potential efficacy in the treatment of a wide range of metabolic and psychiatric disorders18.Neuropeptide Y (NPY), a 36-amino acid (aa) peptide originally isolated from the porcine brain22. Since discovery of the first invertebrate NPF in the tapeworm Monieza expansa23, several NPF orthologs that typically display an RPRF-NH2 C-terminal sequence and a length ranging from 36 to 40 aa have been identified from diverse invertebrate phyla20. However, functional studies for NPF signaling systems with NPFRs and their related receptors have mainly focused on model organisms, especially Drosophila and Caenorhabditis21. Invertebrate NPF systems, including sNPF, seem to play crucial roles in the coordination of feeding, energy metabolism, circadian rhythm, and reproduction26. Recent findings offer new insights into the roles of the Drosophila NPF signaling pathway in male courtship and germline stem cell proliferation28.Invertebrate NPF, an ortholog of human NPY terminating in a Phe-amide, and NPF-related NPs  exert their effects on target cells by binding to and activating specific types of receptors, leading to changes in the activity of downstream effectors29. Although the first functional invertebrate NPFR was characterized from the brain of the snail Lymnaea stagnalis30, there is limited and controversial information on the in vivo effects of molluscan NPFs. Injection of NPF increased the filtration rates of clams (Ruditapes philippinarum)31, whereas Aplysia californica NPF was found to reduce food intake in a dose-dependent manner32. Further, administration of L. stagnalis NPF to the snail did not have short-term effects on food intake, but reduced growth and reproductive performance34. To fully explain these elusive NPF functions, it is essential to investigate the distribution, spatiotemporal expression pattern, and functional characterization of NPF signaling systems in non-model mollusks. Since the L. stagnalis NPFR is the only known functional receptor interacting with molluscan NPF identified to date30, characterization of additional receptors is needed to better understand the possible roles of NPF in mollusks.Mollusca is the second-largest phylum of living animals, with an estimated 85,000 extant species of mollusks comprising approximately 23% of all named marine animals35. However, environmental and farm stress imposes changes to abalone physiology and metabolism, such as reductions in juvenile growth rates and spawning, threatening the global and sustainable aquaculture industry of abalone36. To better understand the reproductive mechanism in the Pacific abalone Haliotis discus hannai (Hdh), we recently analyzed the neural ganglia transcriptome and peptidome associated with sex, growing stage, and sexual maturation of this species38. Given that NPF/Y signaling is known to regulate food intake in both vertebrates and invertebrates20, we hypothesized that Hdh-NPF regulates food intake in Pacific abalone. We here report the identification and characterization of a functional Hdh-NPF signaling system. The novel molluscan Hdh-NPFR can provide further insights into the evolution and function of NPF/Y receptors across bilaterian organisms.Abalone  farms provide high-quality seafood for humans. Recently, farm production of this species has increased from negligible quantities, with the vast majority being produced in China and South Korea at 87% and 10% respectively, according to worldwide production recordsNPF transcripts (Hdh-NPF) were 621\u00a0bp and 647\u00a0bp, including a 5\u2032-untranslated region (UTR), 3\u2032-UTR, and poly(A) tail . The nucleotide sequence identities between two Hdh-NPF genes of exon 2, 3, and 4 were 91%, 100%, and 96%, respectively. The first and second exons (E1 and E2) of the shorter 7.2\u00a0kb-long gene commonly encoded 5\u2032-UTRs, signal sequences, and the majority of Hdh-NPF peptide regions in two prepro-Hdh-NPF precursors, whereas the last two exons (E4 and E4\u2032) of the 8.7\u00a0kb-long gene were alternatively spliced for the two transcripts  and human NPYRs. Among them, we named the NPFR with the highest aa sequence identity (57.4%) to L.sta_NPFR as Hdh-NPFR (373 aa) and the others were designated Hdh-NPFR-like-1 (419 aa), -2 (377 aa), and -3 (390 aa) in the order of sequence similarity (27.4\u201322.9%). The abalone receptors had one N-terminal extracellular domain (ECD), seven trans-membrane domains (TMDs), three extracellular and intracellular loops (ECLs and ICLs), and one C-terminal intracellular domain (ICD). We also determined two potential N-glycosylation sites in the ECD and a characteristic E/DRY/F sequence of rhodopsin-like GPCR in the second ICL of Hdh-NPFR  in the starfish Asterias rubens. Two consensus protein kinase C (PKC) phosphorylation sequences (R/K-X-S/T) were observed in the C-terminal ICD of Hdh-NPFR as a similar feature with the consensus PKC and PKA phosphorylation sites (R-X-S/T or R-R/K-X-S/T) in the C-terminal ICD of A.rub_sNPF/PrRP-R, Drosophila NPFR (D.mel_NPFR), and human NPYR1/2. Comparison of the exon\u2013intron structures and coding sequences (CDS) of Hdh-NPFR and Hdh-NPFR-like genes with those of L.sta_GPCR105, A.rub_sNPF/PrRP-R, and human and chicken NPYR1/2/5 genes showed that Hdh-NPFR and Hdh-NPFR-like genes were more similar to A.rub_sNPF/PrRP-R and vertebrate NPYR2 than to NPYR1 and NPYR5, in terms of an intron insertion in the CDS in NPYR1 and a longer third ICL in the CDS of NPYR5  were identified by a BLAST search with L.sta_NPFR, echinoderm sNPF/PrRP-Rs, and vertebrate NPYR2/7 with a relatively lower bootstrap value (45%); Hdh-NPFR-like-1 and Hdh-NPFR-like-2/-3 receptors were positioned in the subclades composed of lophotrochozoan and ecdysozoan NPF/Y receptors, and another lophotrochozoan NPFR-like receptors, respectively. All the Hdh-NPFR-like-1/2/3 showed a sister relationship with the subclade composed of vertebrate NPYR1/4/5/8.To investigate relationships of the Hdh-NPFR and Hdh-NPFR-like receptors with other bilaterian NPF/Y and related receptors, we performed a phylogenetic analysis using a sequence database with bilaterian NPF/NPY/sNPF receptors and other closely related receptors . This analysis revealed that the Hdh receptors were positioned in a large bilaterian NPF/Y receptor superfamily, with a bootstrap support of 71% Fig.\u00a0. More spExpression of hemagglutinin (HA)-tagged Hdh-NPFRs in human embryonic kidney 293 (HEK293) cells was verified by immunocytochemistry (ICC) with an anti-HA antibody. ICC analysis revealed that Hdh-NPFR and Hdh-NPFR-like receptors were mainly expressed in the cell membranes, although immunoreactive signals were also detected in the cytoplasm -K1 cells transfected with Hdh-NPFR- and aequorin-expressing plasmids with or without the promiscuous G\u03b115 plasmid. If Hdh-NPFR activation leads to Ca2+ increase without the promiscuous G\u03b115, this indicates that the Hdh-NPFR couples to the endogenous Gq protein to activate the phospholipase C (PLC)/inositol trisphosphate/PKC/Ca2+ release pathway in the CHO-K1 cells. Hdh-NPFR-transfected cells generated robust luminescence responses to Hdh-NPF in a dose-dependent manner, regardless of the presence of G\u03b115  or cAMP response element (CRE-Luc) were applied to determine ERK/MAPK activity, Ca\u03b115 Fig.\u00a0a. Howeve\u03b115 Fig.\u00a0, indicat2+ mobilization response in the Hdh-NPFR-transfected cells , pleuro-pedal ganglia (PPG), ovary, gills, intestine, and hepatopancreas of female abalone. The prepro-Hdh-NPF transcript was dominantly expressed in the CG, with a significantly higher level found in sexually mature females than in immature females ] showed a significant increase in food consumption  compared with that observed following saline injection , resulted in loss of agonist-induced functions, including Ca2+ mobilization, ERK activation, and receptor internalization49. PKC phosphorylation sites in the C-terminal ICD were similarly positioned in the Hdh-NPFR, D.mel_NPFR, A.rub_sNPF/PrRP-R, and human NPYR1/2, whereas consensus glycosylation sites were found within the predicted extracellular N-terminus of all the examined receptors. This suggests that the potential PKC phosphorylation sites in the ICD are not likely involved in the pivotal function of NPF/Y receptors, although the PKC-dependent cascade is clearly involved in activation of Hdh-NPFR as described in further detail below.To date, 43. In addition, we suggest that Hdh-NPFR is most likely similar to vertebrate NPYR2 among the three vertebrate NPYR ancestors, the progenitors of the NPYR1, NPYR2, and NPYR5 subfamilies50. The first line of evidence is the sequence similarity between NPYR2 and Hdh-NPFR based on the BLAST and a phylogenetic analysis. Hdh-NPFR showed connections with the vertebrate NPYR2/7 cluster than with the NPYR1/4/5/8 cluster, although the bootstrap value of the branch was relatively low in this phylogenetic tree. In a previous report, the sequences of deorphanized L.sta_NPFR and D.mel_NPFR were closely related to the vertebrates NPYR2 receptor subtype in a phylogenetic tree based on Clustal W alignment and Jukes-Cantor distance analysis51. The second line of evidence is the exon\u2013intron structure and the aa length of the third ICL of NPF/Y receptors. In human, chicken, and coelacanth fish , a representative of a basal lineage of vertebrates, NPYR1 show a conserved intron interposition in the nucleotide sequence encoding the 5th TMD region, whereas NPYR2 and NPYR5 have no intron sequences in the corresponding regions56. Similarly, Hdh-NPFR and Hdh-NPFR-like receptors had no interposition of introns in the entire open reading frame region. The third ICLs of vertebrate NPYR5 is at least four times longer than the corresponding region of other NPYR subtypes, which is clearly distinguishable from Hdh-NPFR and Hdh-NPFR-like receptors. Interestingly, comparison of the sequence of Hdh-NPFR with the starfish A.rub_sNPF/PrRP-R showed several common features, including the aforementioned predicted E/DRY/F motif in the second ICL, PKC phosphorylation sites in the C-terminal ICD and the exon\u2013intron structure. However, functional studies on Hdh-NPFR and Hdh-NPFR-like receptors are needed for further characterization of molluscan NPFR gene duplication and the ancient complexity of the vertebrate NPY signaling system, since the L.sta_NPFR and A.rub_sNPF/PrRP-R were assumed to be vertebrate NPYR1 and PrRP-R homologs, respectively, based on pharmacological data33.Through the sequence comparison and phylogenetic analysis, here we report that Hdh-NPFR-related receptors are belong to the NPF/Y receptor family distinct from the sNPFR and PrRP-R families in bilaterians. Consistent with this analysis, recent phylogenetic studies with novel NPF/NPY/PrRP/sNPF-related receptors in echinoderms and three lophotrochozoans  have revealed that all the examined molluscan NPFR-like sequences are orthologous to the vertebrate NPY receptorsL.sta_NPFR in a model molluscan species, L. stagnalis30, the characterization of other functional NPF receptors and their downstream signaling pathways in mollusks has long been awaited. Here, we show that Hdh-NPFR is potently activated by the mature Hdh-NPF, and the ligand-activated receptor leads to activation of MAPK/ERK signaling through a Gq-PLC-PKC-dependent cascade and increase of intracellular Ca2+ mobilization, as is the case for L.sta_NPFR, human NPYR2, and chicken NPYR1/2/5/7 receptors55. This provisional pathway is supported by the results of western blot analyses showing that the PKA-specific inhibitor failed to inhibit the phosphorylation of ERK1/2, whereas the PKC inhibitor inhibited ERK1/2 phosphorylation in Hdh-NPFR-expressing cells after Hdh-NPF treatment. Nevertheless, we cannot rule out the possible inhibitory pathway of cAMP/PKA in the liganded Hdh-NPFR signal transduction system. In fact, L.sta_NPFR and vertebrate NPYRs are functionally coupled to more than one secondary messenger system55. The Hdh-NPFR along the cellular membrane translocated to an intracellular compartment in HEK293 cells at 30\u00a0min post treatment of Hdh-NPF, similar to NPY-stimulated human NPYR157, suggesting that Hdh-NPFR transduces Ca2+ responses rapidly through the Gq-PLC-PKC signal cascade. Among the three vertebrate NPYR ancestors, NPYR1, NPYR2, and NPYR5, the most prominent pharmacological feature of NPYR2 is that it can bind N-terminal truncated peptide fragments50. In contrast, truncated Hdh-NPF peptides could not increase the intracellular Ca2+ mobilization in Hdh-NPFR-expressing cells, similar to the response of L.sta_NPFR-expressing cells treated with N-terminal truncated Ls-NPF30. These observations imply that the molluscan NPF signaling systems are not similar to those of vertebrate NPYR2, adding another level of complexity presented by the variation of bilateian NPF/Y system.Since an earlier pioneering study demonstrated that Ls-NPF can activate the cognate receptor 58. These data suggest that the ionic interaction contributed by the acidic E/D residue in TMD6 is critical for recognition of the ligands with a positive charge across bilaterian NPF/NPY receptors, including human NPYR259. Similarly, the R33 residue in human NPY preferentially interacts with D292  in human NPYR260. In this context, Hdh-NPFR would be a key and ancestral receptor for understanding the evolution of the NPY signaling system. In addition, we observed that the helix region of Hdh-NPF (Q22\u2013Y31) contacts the ECL3 region of Hdh-NPFR (S288\u2013K293) connecting TMD6 and TMD7 outside the binding pocket, which can explain the stronger energy of Hdh-NPF binding than that of human NPY binding to Hdh-NPFR. Taken together, the docking model presented plausible binding mode of Hdh-NPF with the conserved key interactions.To identify the receptor residues that are important for Hdh-NPF binding, we performed a docking simulation to predict the binding mode of Hdh-NPF peptide and estimated the critical interactions in the Hdh-NPFR binding pocket. The docking model suggested that the C-terminal I33\u2013F39 residues of Hdh-NPF are the major sites interacting with the binding pocket of Hdh-NPFR, under the reasonable assumption that R38 of Hdh-NPF interacts with E284 of TMD6 in the Hdh-NPFR extracellular region as the salt bridge formation between R35 and D287 in human NPYR161 revealed that injection of NPY into the hypothalamic paraventricular nucleus stimulates food intake in rats, the role of NPY/F signaling in regulation of feeding has been widely demonstrated in diverse vertebrate and invertebrate phyla20. In this study, a high dose of Hdh-NPF peptide injection could increase seaweed consumption, whereas injection of dsRNA for Hdh-NPF mRNA remarkably decreased food intake in Pacific abalone. These findings suggest that NPF production induces feeding behavior with a consequent increase of food intake, as observed across bilaterian species. In fact, feeding rhythms, ingestion rate, and digestive enzyme activities were positively correlated with the gene expression levels of Hdh-NPF in Pacific abalone62 and injection of NPF peptide was reported to significantly increase the filter feeding rate in the molluscan bivalve R. philippinarum31. In contrast, Ls-NPF showed no short-term effect on food intake in L. stagnalis34 and its role seems to be quite different from those of abalone NPF and mammalian NPY10. Instead, a significant increase in food intake started at 9\u00a0days after implantation of Ls-NPF34, suggesting that NPF is a long-term effector of food consumption in L. stagnalis. The possible roles of NPF in these regulatory mechanisms underlying food intake should be further determined in diverse molluscan species.Since Stanley and LeibowitzIn conclusion, Hdh-NPF, Hdh-NPFR, and Hdh-NPFR-like receptors are eligible molecules to extend our understanding of the evolution and diversity of the metazoan NPF/Y-mediated signaling system. This work can contribute relevant information for practical applied in monitoring the physiological homeostasis and metabolism of Pacific abalone as an important economic species that is under threat due to extensive environmental stress from farming to meet the high market demand. Further elucidation of NPs with their cognate receptors in mollusks can provide opportunities to search endogenous NPs for uncharacterized human GPCRs, and to develop pharmaco-therapeutic ligands for human NP signaling systems.H. discus hannai, were previously determined and were obtained from transcriptome databases38. Functional annotations were conducted by comparing the sequences against those in public databases, including the National Center for Biotechnology Information (NCBI) BLASTp program. Sequence alignments for bilaterian representative NPF/Y peptides  was used to highlight conserved amino acids. Sequence alignments for Hdh-NPFR-related receptor sequences were performed using CLC Genomics Workbench software. The prediction of transmembrane helices in Hdh-NPFR-related receptors was performed using the latest version of the TMHMM program64. The N-linked glycosylation and intracellular phosphorylation sites were predicted by the NetNGlyc and NetPhos servers, respectively . The exon\u2013intron boundaries were predicted by BLAST of NCBI Genome Workbench.Transcripts for the Hdh-NPF precursors (NCBI GenBank accession numbers MZ027150\u2013MZ027151), Hdh-NPFR (MZ014382), and Hdh-NPFR-like receptor sequences (MZ014383\u2013MZ014385) of Pacific abalone, 22 and the NCBI nr repository. In total, 30 prepro-hormones and 83 receptors were aligned using MUSCLE in the online tool NGPhylogeny 65 and automatically trimmed using trimAL in the online tool NGPhylogeny66. The trimming contained a total of 39 and 270 residues for prepro-hormones and receptors, respectively, that were used to generate the maximum likelihood tree using W-IQ server v1.6.1267. The substitution models, PMB\u2009+\u2009I\u2009+\u2009G4 for prepro-hormones and LG\u2009+\u2009F\u2009+\u2009I\u2009+\u2009G4 for receptors, and the ultrafast bootstrap approximation approach and SH-aLRT 1000 replicates were used. Phylogenetic trees were visualized, using the free software package FigTree v1.4.3 by A. Rambaut at http://tree.bio.ed.ac.uk/software/figtree/.To generate phylogenetic trees, aa sequences of bilaterian NPF/Y and sNPF prepro-hormones  was purchased from a local dealer . Total RNA was extracted from the PPG using the RNeasy Mini kit  and first-strand cDNA was synthesized using PrimeScript RT reagent kit with gDNA Eraser  according to the manufacturer instructions. Polymerase chain reaction (PCR) was performed using the synthesized PPG cDNA as a template, PrimeSTAR HS DNA polymerase (Takara), and oligo primer sets  and NeuroPred  servers along with previous alignment data for NPF sequences19. Peptides for abalone Hdh-NPF and its truncated/mixed NPFs with NPY were synthesized by Anygen  with a purity of >\u200995% analyzed by high-performance liquid chromatography . The cells were transfected with a luciferase reporter plasmid of CRE-Luc68 or SRE-Luc69 using a polyethyleneimine transfection reagent , along with pcDNA3-HA plasmids containing Hdh-NPFR-related receptors, human NPYR1 and NPYR2 , and a pRSV-\u03b2-galactosidase expression plasmid as an internal control (100\u00a0ng of each plasmid/well) as previously reported68. After 3\u00a0h of transfection, the culture medium was replaced by new DMEM with 1% P/S and 10% FBS, and the HEK293 cells were further cultured for 30\u00a0h and then maintained in DMEM without FBS for 16\u00a0h. Finally, the HEK293 cells were treated with peptides, forskolin (Sigma-Aldrich), 12-O-tetradecanoylphorbol-13-acetate , or the same volume of peptide-free medium as a vehicle for 6\u00a0h. The cells were lysed with a luciferase cell culture lysis buffer  and luciferase activities were analyzed using a microplate-luminometer  and normalized by the \u03b2-galactosidase values detected by an absorbance microplate reader  at 405\u00a0nm.HEK293 cells were cultured in Dulbecco\u2019s modified Eagle medium  containing 1% penicillin/streptomycin  and 10% fetal bovine serum . Sixteen hours before transfection, cultured HEK293 cells were seeded into 24-well plates (5\u2009\u00d7\u2009102. Cells grown in a monolayer to 60% confluency in 100\u00a0mm dishes (1.5\u2009\u00d7\u2009106 cells) were transiently transfected with plasmids using FuGENE6 transfection reagent (Promega), according to the manufacturer\u2019s recommendations. In brief, the transfection medium was prepared by combining 600\u00a0\u03bcl of DMEM-F12 with 2\u00a0\u03bcg of pcDNA3-HA-Hdh-NPFR plasmid together with 2\u00a0\u03bcg of a Ca2+ reporter aequorin plasmid with or without the promiscuous G\u03b115 expression plasmid  with 18\u00a0\u03bcl of FuGENE6 in a 5\u00a0ml polystyrene tube and incubated for 25\u00a0min at room temperature. The transfection mixture was added to CHO-K1 cells in 10\u00a0ml of fresh FBS-free medium and the cells were incubated at 37\u00a0\u00b0C in 5% CO2 overnight. On the day of the assay, the cells were detached from the culture dish by incubating with 3\u00a0ml of 5\u00a0mM EDTA in phosphate-buffered saline  for 5\u00a0min. The cells were washed in 10\u00a0ml of phenol red-free DMEM/F-12 with 0.1% bovine serum albumin (BSA) and 1% P/S (DMEM-prf), and collected by brief centrifugation. Probenecid  and coelenterazine  were added to resuspended CHO-K1 cells in 5\u00a0ml of DMEM-prf medium and the cells were gently agitated for 2.5\u00a0h using a magnetic stirrer at room temperature shielded from light. The cell suspension was diluted four times with DMEM-prf and incubated for a further 30\u00a0min. Just before the Ca2+ mobilization assay, peptide solutions were prepared in DMEM-prf and 50\u00a0\u03bcl aliquots were dispensed into 3 or 4 wells/peptide on 96-well microplates. DMEM-prf was used as a negative control. While stirring gently, 50\u00a0\u03bcl of the cell suspension was injected into a luminescence microplate reader (Berthold Tech.) using an automated injector unit. Luminescence was recorded for 20\u00a0s and for a further 10\u00a0s after injection of DMEM-prf including 0.2% Triton X-100 to measure the total Ca2+ response. The average luminescence from multiple replicate wells for a single concentration was calculated and normalized to the largest receptor response after subtraction of background values obtained from negative controls. Dose\u2013response curves and EC50 values were obtained using Sigma Plot v.13 .CHO-K1 cells were maintained in DMEM-F12 Nutrient Mixture Ham supplemented with 10% FBS (Welgene) at 37\u00a0\u00b0C in 5% COd-lysine hydrobromide (Sigma-Aldrich)-coated coverslips, and pcDNA3-HA-Hdh-NPFR and related receptor constructs (300\u00a0ng each) were transfected as described for the luciferase reporter assays. After 30\u00a0h of culture, the cells were fixed with 4% paraformaldehyde in PBS , and pretreated with PBS including 1% BSA and 0.1% Tween 20 (PBST buffer) at room temperature for 30\u00a0min. In the case of treatment with Hdh-NPF peptide, the cultured cells were further incubated for up to 30\u00a0min before fixation. The HEK293 cells were treated with a monoclonal HA primary antibody  in PBST at 4\u00a0\u00b0C for 16\u00a0h. After three washes with PBST, the cells were treated with a secondary antibody  in PBST at room temperature for 1\u00a0h in a light-blocked chamber. After three washes with PBST, the cells were mounted with a mounting medium including DAPI , and were monitored with a fluorescence microscope .HEK293 cells were seeded on poly-6 cells/well) and transfected with 3\u00a0\u03bcg of pcDNA3-HA-Hdh-NPFR or pcDNA3 as described for the reporter assays. At 36\u00a0h post-transfection, the transfected cells were treated with Hdh-NPF (10\u20139\u201310\u20137\u00a0M), TPA (10\u20137\u00a0M), or the same volume of peptide-free medium as a vehicle for 10\u00a0min. After washing twice with PBS, the cells were lysed with RIPA buffer  including protease inhibitor cocktail and PhosSTOP  according to the manufacturer\u2019s recommendation. To examine signaling pathways, Hdh-NPFR-transfected cells were pre-incubated with serum-free DMEM for 6\u00a0h at 37\u00a0\u00b0C and exposed to a PKA inhibitor  or PKC inhibitor  for a further 60\u00a0min. After replacing the medium with fresh serum-free DMEM, the cells were exposed to Hdh-NPF peptide  for 5\u00a0min at 37\u00a0\u00b0C. The cell lysates were separated by 10% SDS\u2013polyacrylamide gel electrophoresis and subsequently transferred to nitrocellulose membranes . After blocking with TBST buffer  containing 5% skim milk  at room temperature for 1\u00a0h, the membranes were incubated with a monoclonal mouse p44/42 MAPK or rabbit phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) primary antibody  in TBST including 2.5% skim milk at 4\u00a0\u00b0C for 16\u00a0h. After three washes with TBST, the membranes were treated with goat anti-mouse IgG-HRP or goat anti-rabbit IgG-HRP secondary antibody  in TBST including 2.5% skim milk at room temperature for 2\u00a0h. The membranes were washed with TBST buffer twice and finally incubated with an enhanced chemiluminescence western blotting substrate reagent (Thermo Fisher Scientific) at room temperature for 5\u00a0min. The reactive bands were monitored using C-Digit 3600 Bolt Scanner .HEK293 cells were seeded into 6-well plates  was considered as the final Hdh-NPF structure model. The 3D structure of Hdh-NPFR was modeled using SWISS-MODEL71. The best template for homology modeling, 5zbh (NPYR1 in T4 lysozyme), showed 31% sequence identity with Hdh-NPFR. The N- and C-terminal partial sequences were not modeled because none of the template structures covered the sequences and the N-terminal sequence in the ectodomain was disordered in the template structure.The three-dimensional (3D) structure of the 39-residue-long Hdh-NPF peptide was first modeled using PEP-FOLD372. The docking simulation consisted of an initial energy minimization, fast relax  application with distance constraints, and fast relax without a constraint. The distance constraint was defined between R35 in Hdh-NPF and E287 (6.59) in Hdh-NPFR given the results of recent structural studies on NPY and the cognate receptors59. The constraint ensured that the Hdh-NPF peptide is inserted into the binding pocket. The final relax application fully refined the complex structure without any constraints to check if the docked Hdh-NPF peptide structure is stably maintained in the binding pocket. Among 500 docking trials, the docking conformation with the lowest binding energy was considered as the final NPF-NPFR complex model (\u0394\u0394G \u2212\u200951.55 Rosetta Energy Unit by the REF2015 energy function).The peptide and receptor model structures were used to perform docking simulation by RosettaScriptH. discuss hannai  and immediately frozen in liquid nitrogen before storage at \u2212\u200980\u00a0\u00b0C. The reproductive stage of abalone gonads was classified according to a previous study58. Total RNA was extracted using the RNeasy Mini kit (Qiagen) and 1\u00a0\u03bcg of RNA was reverse-transcribed to first-strand cDNA using the PrimeScript RT reagent kit (Takara). The ribosomal protein L-5 gene (Hdh-RPL5) was used as an internal reference for RT-qPCR as previously described73. Gene-specific primers for amplifying the Hdh-NPF precursor and Hdh-NPFR genes are listed in Table The CG, PPG, ovary, gills, intestine, and hepatopancreas were dissected from adult female Saccharina japonica) before use in experiments. To reduce consumption rate bias based on individual variation, the abalone were starved for 48\u00a0h, placed in individual containers, and refed for 24\u00a0h before the experiment. On the day of the experiment, kelp pieces were divided into two equal parts, blotted, and weighed to obtain the wet mass (g) before and after the feeding period. To correct for autogenic changes in kelp mass over time, one part was secured to the rim of the treatment cage and the other was secured to the control cage without abalone. Abalone were weighed prior to each assay (n\u2009=\u200911 per group) and 350\u00a0\u03bcl of mollusk saline  including Hdh-NPF (0.25 or 2.5\u00a0\u03bcg/g BW) was injected into the adduct muscle sinus using a 26-gauge needle. Control abalone were injected with the same volume of mollusk saline. Injected abalone were individually placed in a cage (15.5\u2009\u00d7\u200911\u2009\u00d7\u20096.5\u00a0cm) with flow-through seawater and supplied with seawater-immersed kelp equivalent to 7% of the BW. Food intake was assessed at 24\u00a0h post-injection as follows: Consumption (W)\u2009=\u2009[Wi\u2009\u00d7\u2009(WCf/WCi)\u2009\u2212\u2009Wf], where Wi is the initial wet kelp weight, Wf is the remaining wet kelp weight, and WC is an autogenic control to determine the permeation of water into kelp during the feeding time. Consumption values were standardized for abalone BW to 100\u00a0g.Pacific abalone (33.28\u2009\u00b1\u20090.81\u00a0g BW) were maintained in a flow-through seawater aquarium  for 2\u00a0weeks, and fed ad libitum on kelp (Hdh-NPF amplicons as templates for the synthesis of dsRNA-Hdh-NPF using the T7 Ribomax Express RNAi system (Promega). As described in the preceding section, Pacific abalone  were starved, refed, and injected with 120\u00a0\u03bcl of 1.38\u00a0\u03bcg/\u03bcl dsRNA-Hdh-NPF or the same volume of mollusk saline into the adduct muscle sinus (n\u2009=\u200910 per group). The quantity of 165\u00a0\u03bcg of dsRNA-Hdh-NPF, corresponding to a mean concentration of 10\u00a0\u03bcg of dsRNA per gram of abalone BW without shell weight, was within the range of dsRNA quantities injected into other invertebrates (4\u20138\u00a0\u03bcg for shrimp Litopenaeus vannamei and 20\u00a0\u03bcg for oyster Crassostrea gigas per gram BW)75. At 72\u00a0h post-injection, abalone were supplied with seawater-immersed kelp and food consumption was measured for 24\u00a0h. Immediately after the feeding assay, CG tissues were dissected from abalone, frozen in liquid nitrogen, and then stored at \u2212\u200980\u00a0\u00b0C until RNA extraction. Total RNA extraction, cDNA synthesis, and RT-qPCR for Hdh-NPF, Hdh-GnRH, and Hdh-APGWa transcripts were conducted as described above, except for the primers used (Table 76.A 412-bp cDNA fragment (nucleotides 48\u2013460) of Hdh-NPF precursor (NCBI accession numbers MZ027150\u2013MZ027151) was amplified by PCR using gene-specific primers with a T7 promoter tag (Table Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4."}
+{"text": "While high-fat diet feeding induced obesity for huCETP mice and their wild-type littermates lacking CETP expression, insulin sensitivity was higher for female huCETP mice than for their wild-type littermates. There was no difference in insulin sensitivity for male huCETP mice vs. littermates. The increased insulin sensitivity in females was largely caused by the better insulin-mediated suppression of hepatic glucose production. In huCETP females, CETP in the circulation decreased HDL-cholesterol content and increased liver cholesterol uptake and liver cholesterol and oxysterol contents, which was associated with the upregulation of LXR target genes in long-chain polyunsaturated fatty acid biosynthesis and PPAR\u03b1 target genes in fatty acid \u03b2-oxidation in the liver. The upregulated fatty acid \u03b2-oxidation may account for the improved fatty liver and liver insulin action in female huCETP mice. This study provides further evidence that CETP has beneficial physiological roles in the metabolic adaptation to nutrient excess by promoting liver fatty acid oxidation and hepatic insulin sensitivity, particularly for females.Mounting evidence has shown that CETP has important physiological roles in adapting to chronic nutrient excess, specifically, to protect against diet-induced insulin resistance. However, the underlying mechanisms for the protective roles of CETP in metabolism are not yet clear. Mice naturally lack CETP expression. We used transgenic mice with a human CETP minigene (huCETP) controlled by its natural flanking region to further understand CETP-related physiology in response to obesity. Female huCETP mice and their wild-type littermates were fed a high-fat diet for 6 months. Blood lipid profile and liver lipid metabolism were studied. Insulin sensitivity was analyzed with euglycemic-hyperinsulinemic clamp studies combined with  In humans, cholesteryl ester transfer protein (CETP) shuttles cholesteryl esters from HDL to apoB-containing particles (VLDL and LDL) and transfers triglycerides (TG) from apoB-containing particles into HDL in the circulation. CETP-mediated shuttling of cholesteryl esters from HDL to apoB-containing particles is part of the reverse cholesterol transport process where cholesterol is delivered to the liver for secretion as bile acids . CETP-me\u2013/\u2013Ldlr or \u2013/\u2013apoE mice, CETP expression increases atherosclerotic burden because of the redistribution of cholesterol in lipoprotein particles in the setting of impaired LDL clearance  controlled by its natural flanking sequences. In contrast to transgenic mice overexpressing the simian CETP driven by a constitutive promoter, mice expressing huCETP have blood CETP levels and a tissue CETP expression pattern similar to that seen in humans . With th\u00b0C with a 12-h-light/12-h-dark cycle. Transgenic simian CETP mice (sCETP) mice were from Jackson Laboratory (C57BL/6-Tg(CETP)1Pnu/J, strain 001929) as we described before  on a C57BL/6J genetic background were purchased from the Jackson Laboratory (B6.CBA Tg(CETP) 5203Tall/J). Transgenic mice were crossed with C57BL/6J mice (strain 000664) and were housed at 23d before . Transged before . After 5d before , 2018a. Mouse blood samples were collected after 5 h of fasting before the insulin clamp study. Fasting serum cholesterol and triglyceride levels were determined using Infinity\u2122 Cholesterol and Triglycerides reagents from Thermo Scientific according to the manufacturer\u2019s instructions (Cat# TR13421 and TR22421).Blood inflammatory factors interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) and other chemistry parameters were determined by the Vanderbilt Hormone Assay Core laboratory.Blood insulin concentrations were determined using Rat/Mouse Insulin ELISA Kit from Millipore following the manufacture\u2019s instruction (Cat#EZRMI-13K).The second cohort of mice was prepared with a high-fat diet feeding for measuring blood ketone body levels. Blood ketone body levels at different time points of fasting were determined using the Precision Xtra Blood Glucose and Ketone Monitoring System .The third cohort of mice was prepared with a high-fat diet and sacrificed after 5 h of fasting for determining liver TG content during fasting.n = 6) and age-matched female simian CETP mice (n = 4) and female wild-type mice (n = 6) fed a chow diet were used for blood CETP activity assay. Human blood samples for the CETP activity assay were purchased fresh frozen plasma and de-identified samples from the Vanderbilt Lipid Clinic and Laboratory.Blood CETP activity was measured using a cell-free assay according to the manufacturer\u2019s protocol  as we reported previously . FastingWe used size exclusion chromatography (SEC) with a high-performance liquid chromatography (HPLC) system to separate plasma lipoproteins to quantify the distribution of cholesterol. The SEC separation was performed as described with a superose\u2122 6 10/300 GL from GE Healthcare (Cat# 17-5172-01) . FractioAbcam; antibodies for SR-BI (NB400-104) and PDK4 (NBP1-07047) were from Novus, antibodies for DGAT1 (sc-32861) and DGAT2 (sc-668549) were from Santa Cruz; antibodies for AKT (Cat# 9272), pAKT (Cat# 4060), ERK (Cat# 9107), pERK (Cat# 4370), ACC (Cat#3676), and pACC (Cat#3661) were from Cell Signaling. The following primary antibodies were all from Abcam: antibody for LDL receptor (ab30532); anti-SCD1 (ab19862); anti-PGC-1\u03b1 (ab54481); antibodies for ELOVL2 (ab176327) and ELOVL5 (ab205535); antibodies for FADS1 (ab126706) and FADS2 (ab170665), antibody for PPAR\u03b1 (ab8934), antibody for ABCA1 (ab66217), antibody for HMGCS2 (ab137043), antibody for MCAD (ab110296), and antibody for CPT1\u03b1 (ab128568). \u03b2-actin was used as loading control for liver tissues and detected with either rabbit anti-actin (I-19) antibody (sc-1616) from Santa Cruz or with mouse anti-human actin antibody (MCA5775GA) from Bio-Rad. \u03b1-Tubulin was used as loading control for muscle tissues and detected with an antibody from Abcam (ab4074). All primary antibodies were diluted at 1:1,000, except where otherwise noted, and incubated at 4\u00b0C overnight. All secondary antibodies were from LI-COR Odyssey  and diluted at 1:10,000 or 1:15,000 and incubated at room temperature for 1 h. Images were acquired using an LI-COR Odyssey infrared imaging system (LI-COR Biotechnology). Blot density was quantified and analyzed with Image J and plotted with Prism.Western blotting for liver proteins was performed as reported previously . The priLiver lipid content including triglycerides, cholesterol, diacylglycerol, and fatty acid compositions in triglycerides and phospholipids from the 1st cohort mice were analyzed at the Vanderbilt University Medical Center Lipid Core.v/v), and then isolated and analyzed with Infinity\u2122 Triglycerides reagents.Liver TG content during fasting from the 3rd cohort mice were determined with the method we reported previously . BrieflyTotal RNA was isolated from approximately 50 mg of snap-frozen tissues using a RNeasy mini kit (Qiagen). cDNA was synthesized using the Quanta qScript cDNA synthesis kit (Quantabio). Real-time PCR was performed using TaqMan\u2122 Fast Universal PCR Master Mix (2X) and TaqMan Gene Expression assays  on the QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems).d7-ketocholesterol (10 \u03bcM) internal standard, 400 \u03bcL 0.9% NaCl, and 600 \u03bcL Folch solution  were added to 100 \u03bcL of each sample. The CHCl3 layer was collected, dried, and derivatized by the addition of 100 \u03bcL of a fresh-made N,N-dimethylglycine (DMG) solution . After 30 min agitation at room temperature, the resulting solution was evaporated in Speed Vacuum Concentrator and reconstituted in 100 \u03bcL of methanol  formic acid, 1 mM NH4OAc) for 10 min runtime at a flow rate of 400 \u03bcL/min. A TSQ Quantum Ultra mass spectrometer (Thermo Fisher Scientific) was used for MS detections, and data were acquired with a Finnigan Xcalibur software package. Selected reaction monitoring (SRM) of the DMG derivatives was acquired in the positive ion mode using electrospray ionization (ESI). The SRM transition of precursor to product ion included m/z 493.4 \u2192 390.4 for DMG-d7-7keto-cholesterol, 573.5 \u2192 470.5 for DMG-4\u03b2OH-cholesterol, 486.3 \u2192 383.3 for DMG-7keto-cholesterol, 488.3 \u2192 367.3 for DMG-25OH-cholesterol, 573.5 \u2192 367.4 for DMG-22\u03b2OH-cholesterol, 573.3 \u2192 470.3 DMG-27OH-cholesterol. The response factors of other oxysterols were determined against d7-7keto-cholesterol. Final sterol numbers are reported as nmol/mg tissues.Liver samples were weighed (50\u2013100 mg) and lysed in 0.5 ml tissue lysis buffer containing 0.9%NaCl, 50 mM HEPES, 0.01% TritonX100, 0.1% SDS, protease inhibitors and 10 \u03bcL antioxidant solution (2.5 mg TPP and 1.0 mg BHT in 1 mL EtOH). 10 \u03bcL methanol . Ten mict-test with Welch\u2019s correction as appropriate. Significance was flagged by P < 0.05. Data were presented as means \u00b1 SD unless otherwise indicated.All measurements passed D\u2019Agostino and Pearson omnibus normality test . Differences between groups were determined by ANOVA followed by multiple comparison tests or by Student\u2019s Mice carrying the human CETP minigene (huCETP) have a tissue expression pattern of CETP that is similar to humans . To bettMice expressing the human CETP minigene and their WT littermates were fed a high-fat diet (HFD) for 6 months. The preliminary data showed that there were no significant differences between male huCETP and WT male littermates in glucose infusion rate during euglycemic-hyperinsulinemic clamps . In addiHFD-feeding increased body weight and adiposity in both female huCETP and WT female littermates, and changes in body weight were not significantly affected by huCETP expression . There w473Ser in insulin signaling in the liver was modestly increased in huCETP mice in comparison to their WT littermates  compared to WT littermates , which wFatty liver is a known contributor to insulin resistance . Thus, wTo understand whether the huCETP expression would modify pathways that promote liver lipid uptake, we investigated liver SR-BI and LDLR protein levels. The expression of huCETP increased liver SR-BI protein levels and did not affect liver LDLR protein levels . These rCpt1\u03b1 and Pdk4 are target genes of PPAR\u03b1-activated transcription. However, the liver PPAR\u03b1 protein amounts were not altered by the transgenic expression of huCETP, neither were the PGC-1\u03b1 protein amounts  transports fatty acids into mitochondria for \u03b2-oxidation in the liver . Liver C amounts . We next amounts . These dWith regard to TG synthesis pathways, liver acetyl-CoA carboxylase (ACC) and its phosphorylated form pACC were not significantly altered by the expression of human CETP minigene . DiacylgTo understand the mechanisms that drive hepatic fatty acid \u03b2-oxidation in huCETP transgenic mice, we further explored the ligand-activators for PPAR\u03b1 signaling since both liver PPAR\u03b1 and PGC-1\u03b1 protein levels were not changed. Long-chain polyunsaturated fatty acids (LC-PUFAs) are important ligands to activate PPAR\u03b1 . Thus, wFads1/2) was generally higher in huCETP mouse liver, with a 165% higher level of Fads2 mRNA than WT littermates (We then examined liver protein levels of enzymes that regulate elongation of very-long-chain fatty acids (ELOVL) and fatty acid desaturases (FADS) in LC-PUFA biosynthesis pathways . ELOVL2/termates . Proteintermates . These dHmgcs2 and Acadm are PPAR\u03b1 target genes, HMGCS2 is a regulatory enzyme for ketogenesis and ACADM is a regulatory enzyme for fatty acid oxidation  \u03b1 and \u03b2. We found that the transgenic expression of human CETP minigene increased levels of 4\u03b2OH-cholesterol in the liver but did not lead to a significant change in other measured oxysterol levels . We werexidation . Our resIn this study, we show that female transgenic mice expressing the human CETP minigene containing its natural regulatory elements are protected from diet-induced insulin resistance. With tracers, we show that this improved insulin sensitivity was attributed to the improved insulin-mediated suppression of hepatic glucose production (EndoRa) during the euglycemic-hyperinsulinemic clamp. Improved liver insulin signaling pathways were also associated with decreased liver TG content in huCETP transgenic mice. Furthermore, expression of the human CETP minigene increased liver LC-PUFA levels and stimulated hepatic fatty acid \u03b2-oxidation pathways, which is likely responsible for the limited liver TG content and improved whole-body insulin sensitivity during high-fat diet feeding.Cpt1a and Cpt2 gene expression was accompanied by elevated mRNAs of Ppar\u03b1, Acox, and Acadm, genes that govern fatty acid oxidation in the liver (n = 10/group), we did not observe sex differences in blood CETP activity but observed a reverse association between plasma TG levels and CETP activities in men only (Expression of the human CETP minigene limited diet-induced fatty liver by promoting liver fatty acid \u03b2-oxidation in female mice. The upregulation of fatty acid \u03b2-oxidation signaling in the liver has been reported in mice overexpressing the simian CETP gene . In simihe liver . In the he liver . Reciprohe liver . These she liver . Studieshe liver . With a men only .With the huCETP mouse model, we demonstrate that CETP expression increased hepatic fatty acid \u03b2-oxidation, which is likely mediated by PPAR\u03b1 pathway. It has been shown that the activation of PPAR\u03b1 improves steatosis, inflammation, and fibrosis in rodent models of non-alcoholic fatty liver by promoting peroxisomal and mitochondrial \u03b2-oxidation . Unlike Cpt1b mRNAs in skeletal muscles was only seen when CETP was abundantly expressed in sCETP mice (The huCETP mouse model in the current study along with previous studies using sCETP mice seems to show that the magnitude of improved insulin signaling relates to the magnitude of increased CETP activity. The improved whole-body insulin sensitivity driven by the suppression of liver G6pase-\u03b1 and hepatic glucose production was seen in both huCETP and simian CETP mice . Though ETP mice . TogetheIn a previous study by Raposo and colleagues, blood glucose levels were not significantly different between huCETP and WT mice during either glucose- or insulin-tolerance test . This diThe transgenic expression of human CETP in mice limits liver lipid accumulation and improves insulin resistance during high-fat diet feeding in females. We demonstrate increased fatty acid \u03b2-oxidation in the liver of huCETP mice, which was associated with increased levels of LC-PUFA and the activation of PPAR\u03b1-target genes in fatty acid \u03b2-oxidation signaling pathways. CETP is well-known for its role in modifying lipid distribution between lipoproteins in circulation, which is correlated with the risks for cardiovascular disease. However, the physiological consequences of the CETP-modified changes in lipoprotein lipids in other tissues such as the liver are yet not clear. We have studied CETP biology in models that parallel the eightfold range of CETP activity seen in humans , from ovThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Institutional Animal Care and Use Committee at Vanderbilt University.LZ wrote the manuscript and researched data. JA, SC, TL, YDP, BL, KF, CF, and H-YK researched data and reviewed the manuscript. ML and JS researched data and contributed to discussion and reviewed and edited manuscript. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Enterococcus faecalis  endocarditis and even rarer for such cases complicated by osteomyelitis. Here, we describe the case of a 63-year-old male patient with a recent history of cardiac arrest, a percutaneous endoscopic gastrostomy tube placement, and endotracheal tube placement. He presented with back pain and was found to have osteomyelitis on magnetic resonance imaging and aortic valve vegetations on transthoracic echocardiography (TTE). His blood cultures were positive for E.faecalis. Repeat TTE showed growth in the vegetation, and the patient underwent bioprosthetic aortic valve replacement.Infective endocarditis (IE) is an infection of the heart valves or endocardium, usually due to the spread of infection through the blood. It can cause a varied range of symptoms, from being asymptomatic to reduced heart function, valvular abnormalities, embolization, or death. Enterococci are usually present as normal gut flora but can also cause bacteremia, urinary tract infections, or IE, especially in the elderly population. The source of enterococcal spread in most of the cases is unidentifiable and sometimes associated with the genitourinary tract or damage to the gut mucosa due to trauma, malignancy, and infection, among others. Very few cases have been reported so far on  Enterococcus\u00a0is a genus of gram-positive, facultatively anaerobic bacteria that were previously classified as group D\u00a0Streptococcus, currently a separate genus. Enterococcus faecalis and Enterococcus faecium, which ordinarily inhabit the gastrointestinal, hepatobiliary, and genitourinary flora, are the most prevalent pathogenic subspecies.\u00a0E. faecalis is the third most prevalent cause of IE, accounting for around 5% to 15% of all cases [Infective endocarditis (IE) is one of the less frequent infections, with an annual incidence of five to seven cases per 1,000,000 people.\u00a0ll cases . HoweverE. faecalis. Transthoracic echocardiography (TTE) was performed considering the risk factors, and it showed normal left ventricular ejection fraction (LVEF), a mildly dilated left atrium, moderate aortic regurgitation, moderate-sized vegetation on the aortic valve, and mild to moderate tricuspid regurgitation. Repeat focused TTE showed two 1-cm hypermobile echo densities on the ventricular side of the right aortic valve and a non-coronary cusp, likely vegetation, Figures A 63-year-old male patient with a previous medical history of hypertension, non-obstructive coronary artery disease, cocaine abuse, and a history of cardiac arrest two months back, presented with intractable lower back pain for four days. The patient's previous hospitalization was complicated by bilateral pneumothoraces, chest tube, and percutaneous endoscopic gastrostomy tube placements, followed by discharge after removal of all tubes. This time, the patient had no other symptoms other than back pain,\u00a0such as motor or sensory loss, urinating or defecating difficulties, or saddle anesthesia, but was unable to walk and suffered from severe back pain. Lumbar magnetic resonance imaging showed soft tissue swelling at L5-S1, indicative of osteomyelitis, Figure E. faecalis\u00a0is a type of catalase-negative, gram-positive, facultative anaerobe, often appearing as diplococci in short chains under microscopy [E. faecalis\u00a0IE usually has a high incidence in the aging population, likely due to increased procedures involving the gastrointestinal and urinary tracts, heart valve pathologies, or implanted prosthetic material for the heart\u00a0[E. faecalis\u00a0IE has become a significant infection, considering the increasing frequency of hospital-associated infections and increasing resistance to antibacterial medications\u00a0[IE was first described by William Osler in 1885. It is one of the less frequent infections affecting the heart valves or endocardium, with high mortality and morbidity.\u00a0he heart\u00a0.\u00a0E. faecications\u00a0.Enterococcus-associated osteomyelitis is. Among 462 patients who were treated at the Mayo Clinic from 1969 to 1991 for infected prosthetic joints without IE, only six cases (1.3%) were\u00a0Enterococcus-associated infections. IE cases treated at the Mayo Clinic from 1965 to 1975 totaled 192 patients, out of which 84 (44%) had musculoskeletal complaints, 5 (6%) had possible osteomyelitis, and only one had\u00a0Enterococcus\u00a0as a causative organism [In 2003,\u00a0Nicholas at the Mayo Clinic showed how rare\u00a0E. faecalis\u00a0endocarditis in a patient with Heyde syndrome has been reported\u00a0[The diagnosis of IE requires clinical, laboratory, and imaging examinations. The patient in the present case was diagnosed with IE as two major Duke criteria were positive, i.e., this patient had an episode of\u00a0bloody bowel movement, but the patient did not have endoscopy or colonoscopy. So colorectal carcinoma was one of the differentials that needed to be ruled out. If the patient had aortic stenosis, Heyde syndrome should be considered in differentials since a triad of aortic stenosis, angiodysplasia, and anemia makes Hyde syndrome more likely. A case of\u00a0reported\u00a0. This paE. faecalis\u00a0endocarditis for several years. However, due to the rise of antimicrobial resistance and nephrotoxicity, an alternate therapy of ampicillin and ceftriaxone for four to six weeks is recommended. In 1995, Mainardi et al. initially reported the synergistic effect of ampicillin and ceftriaxone, which was later confirmed by Gavald\u00e0 et al. and Fern\u00e1ndez-Hidalgo as an equally effective and safe alternative, especially in patients with renal failure\u00a0[A combination regimen of aminoglycosides like gentamycin with ampicillin was initially considered and was the standard treatment for\u00a0 failure\u00a0,9.Enterococcus\u00a0is the third leading cause of IE and can present with musculoskeletal complaints. Even though these complaints are common, sufficient evaluations need to be performed to rule out osteomyelitis. Colorectal carcinoma should also be ruled out if the patient presents risk factors or suspicion. Early diagnosis and treatment with dual beta-lactams\u00a0improve survival.IE is a rare and life-threatening cardiac infection with high mortality and morbidity."}
+{"text": "The systemic vascular resistance significantly decreased from 18.85\u200a\u00b1\u200a7.66 mm Hg min L-1 to 14.62\u200a\u00b1\u200a6.13 mm Hg min L-1 30 minutes. The resistance to venous return (VR) significantly decreased from 5.93\u200a\u00b1\u200a4.97 mm Hg min L-1 to 4.46\u200a\u00b1\u200a1.53 mmHg min L-1 3\u200ahours later. The mean systemic filling pressure significantly decreased from 32.71\u200a\u00b1\u200a20.00\u200amm Hg to 28.254\u200a\u00b1\u200a6.09\u200amm Hg 3\u200ahours later.The role of rhBNP on CO was to reduce the peripheral circulation resistance at 30 minutes after rhBNP infusion and to reduce the resistance to VR at 3\u200ahours later.This trial is registered at ChiCTR: ID ChiCTR1900024562. In China, the costs of HF treatments were estimated to be at $5.42 billion and the in-hospital mortality rate was estimated to be at 4.1%. This indicates that HF is a global public health concern.Cardiovascular diseases (CVDs) are major causes of the high mortality rates experienced globally. Heart failure (HF) is the terminal stage of many CVDs characterized by poor prognosis and high mortality rates. The prevalence of HF is estimated to be between 2% to 10% of the global adult population which translates to 38 million people. During the early stages of HF, there is a relative deficit of endogenous BNPs. Therefore, BNP supplements can be used to treat acute decompensated HF. The rhBNP has similar bioactive traits as BNP that influence favorable cardiac effects such as; natriuresis, diuresis, and vasodilation. The rhBNP also inhibits the renin\u2013angiotensin\u2013aldosterone system. Several studies reported that rhBNP has positive therapeutic effects on patients with HF however, it has some side effects such as; decreasing the cardiac preload and afterload with an increase of cardiac output (CO).\u201310 The effect of rhBNP on the systemic venous circulation and its regulation on the CO have not been explored.The treatment of HF was improved through the introduction of drugs such as the angiotensin converting enzyme inhibitors, Angiotensin II Receptor Blockers (ARBs), Aldosterone antagonists and B-adrenergic receptor blocking compounds (\u03b2-blockers). The recombinant human brain natriuretic peptide (rhBNP) was approved by the FDA in 2001 to be used for the treatment of acute decompensated HF. The Brain natriuretic peptide (BNP) is a peptide hormone secreted by ventricular myocytes due to the pressure-volume overloading of ventricles. The systemic veins and venules contain 2/3 of the human body's total blood volume and this contributes to a blood volume reserve. The CO was largely affected by the volume of venous return (VR). Guyton and his co-workers did many experiments to show the relationship between the changes in right atrial pressure (Pra) and the changes in VR, depicted as a venous return curve.,14 The VR is now one of the factors considered when performing hemodynamic monitoring in critical care medicine because it influences the pathophysiology of HF, shock, and vasoactive drugs. There are other factors that affect the VR such as: mean systemic filling pressure (PMSF), right atrial pressure, and the resistance to VR. The PMSF is the upstream driving pressure for VR, defined as the pressure in the vascular system when the circulatory system is at rest. However, PMSF is challenging to quantify. Mass et al 2009 presented a noninvasive way to measure of PMSF by performing 12s inspiratory-hold maneuvers in patients with mechanical ventilation at the bedside. These measurement procedures allowed the calculation of VR parameters from the laboratory to the real patient.During the 19th century, Guyton proposed that the heart function, heart rate and stroke volume are important factors that determine CO.,17 However, the role of rhBNP on the systemic venous circulation in patients with HF remains unclear and there are limited studies that illustrate the effects of rhBNP on CO changes using the Guyton venous return approach. Thus, a retrospective cohort analysis was performed to determine the effects of rhBNP on CO of patients with acute decompensated HF using the Guyton venous return curve approach.Recently, we measured the parameters of VR as a part of hemodynamic monitoring in some of those patients diagnosed with acute decompensated HF. Then some of the above-mentioned patients were treated with rhBNP. The rhBNP is effective in managing acute decompensated HF. It increases the CO and reduces the systemic vascular resistance (SVR) in the pulmonary capillaries.2This study was conducted using the clinical data of patients who were admitted since January 1, 2015 to December 31, 2018 after being approved by the Institutional Ethics Committee of Qingyuan City Hospital. A waiver for the informed consent was obtained since the data was analyzed anonymously. The inclusion criteria was as follows;1.males/females with an age \u226518\u200ayears;2.a history of CVDs and/or HF;3.a diagnosis of acute decompensated HF according to the (ICD-10: I50.1);4.patients who were in the intensive care unit requiring mechanical ventilation in the mode of volume assist\u2013control;5.patients who received hemodynamic monitoring by a PiCCO2 device;6.patients who received diuretics and digoxin before being given an rhBNP intravenous infusion pump and7.patients who had complete data of venous return curve before and after rhBNP intravenous infusion pump.The exclusion criteria included1.Men/women <18\u200ayears of age;2.presence of hypovolemic, cardiogenic or vasodilatory shock, and systolic blood pressure less than 90\u200amm Hg;3.presence of HF with arrhythmias;4.presence of valvular heart disease;5.pregnant or breast-feeding patients;6.patients who had hepatorenal insufficiency  and7.patients who had incomplete information concerning their treatments.2.1This study was retrospective, self-controlled and utilized 1 center. The demographic data in this study included age, gender, heart rate, mean arterial pressure (MAP), body temperature, Acute Physiology and Chronic Health Evaluation score (APACHE II), Sequence Organ Failure Assessment Score (SOFA) and N-terminal B-type natriuretic peptide (NT-proBNP) score. All the data was derived from electronic medical records.\u22121 of body weight), followed by rhBNP infusion of 0.0075\u200a\u03bcg\u00b7kg\u22121 min\u22121 for at least 3\u200ahours. Each patient was given standardized preconditioning included adequate sedation and analgesia. Each patient was mechanically ventilated and had no spontaneous breathing during the measurement process. All patients were placed with a central venous catheter and a femoral artery puncture tube, which was connected with a PICCO2 monitor. The MAP, central venous pressure (CVP), CO), SVR, stroke volume (SV), and stroke volume variation (SVV) were recorded and compared at specific intervals: before rhBNP administration (T 0), 30 minutes after rhBNP administration (T 30\u200aminutes) and 3\u200ahours after rhBNP administration (T 3\u200ahours). The hemodynamic parameters such as CO, SVR, SV, and SVV were obtained using the Transpulmonary Thermodilution Technique (PiCCO TM) from the Pulsion Medical Systems, . The CVP was measured by a venous catheter inserted in the right internal jugular vein by the device of Edwards PX260. The PMSF and the resistance to venous return (RVR) at each interval were calculated as described below.Patients enrolled in this study, were treated with an intravenous loading dose of rhBNP /CO; RVR = (PMSF\u2013CVP)/CO.The protocol for plotting the venous return curve was as follows; Firstly, all the patients was sedated by morphine and midazolam until the Ramsay Sedation Scale was at 5 or 6. The patients were mechanically ventilated in volume assist\u2013control mode with a fraction of inspiration oxygen of 50%, respiratory rate of 16 minutes and a tidal volume of 8\u200amL\u00b7kg-1. Then MAP, CVP, and CO were obtained using 12s inspiratory-hold maneuvers. The above parameters were recorded at the airway plateau pressures of 5, 15, 25, and 35\u200acmH2.2P values <.05 were considered to be statistically significant. The CO and CVP were fitted via linear regression using the least squares method as the venous return curve.Statistical analysis was conducted using the SPSS 22.0 software. The continuous variables were expressed as mean\u200a\u00b1\u200aSD or median with interquartile range. The categorical variables were expressed as frequency  and compared using the Chi-Squared test. The differences in parameters during T 0 and T 30\u200aminutes, T 0 and T 3\u200ahours were compared and analyzed using One-way ANOVA with repeated measures test. 33.1\u22121).We enrolled 149 patients who had been admitted into the intensive care unit of our hospital with a diagnosis of acute left cardiac insufficiency between January 1, 2015 to December 31, 2018. A total of 135 patients were excluded, 34 patients had comorbidities with arrhythmia, 35 patients had a history of heart valve disease, 13 patients were pregnant, 45 patients had liver and renal dysfunction, and 8 patients had incomplete data  30 minutes after rhBNP infusion and CO evidently increased to 4.20\u200a\u00b1\u200a1.19 L\u200amin\u22121 (P\u200a<\u200a.05) 3\u200ahours later. The MAP showed a decreasing trend both at 30 minutes and 3\u200ahours after the rhBNP administration, but with no statistical difference (P\u200a>\u200a.05) (Table P\u200a<\u200a.05) (Table P\u200a>\u200a.05) (Table P\u200a>\u200a.05) . After 3\u200ahours it had not changed its values (Table P\u200a<\u200a.05) (Table P\u200a>\u200a.05) (Table \u22121 to 4.46\u200a\u00b1\u200a1.53\u200amm Hg\u00b7min\u00b7L\u22121 at 3\u200ahours after the rhBNP administration (P\u200a<\u200a.05) /SVR. The VR  equals to the difference between the PMSF and the central venous pressure divided by the resistance to VR; (PMSF-CVP)/RVR. From this study, there were no significant changes in PMSF, CVP or RVR values at 30 minutes after rhBNP administration when compared to the baseline values. This brings a suggestion that there was no significant increase in VR. There was a significant increase in CO at 30 minutes after administration. There were no differences in MAP and CVP as shown by  natriuretic peptide and its binding to type A natriuretic peptide receptor activate guanylyl cyclase (GC). The GC subsequently induces an intracellular rise in cGMP. The cGMP activates the cGMP-dependent protein kinase (PKG) producing the vascular smooth muscle relaxation effects both on arteries and veins. The stressed volume is a decisive factor for PMSF. The stressed and unstressed volume can be converted into each other under certain conditions.,26 In this study, the PMSF values were higher than those of other previous studies.,28 The reason could be the fluid retention and vaso-constriction in patients with acute decompensated HF. From this study, the venous return curve shift to left and PMSF decreased after the rhBNP administration. The decrease in PMSF is the result of the stressed volume shift to the unstressed volume, due to the vasodilatory effects of rhBNP. At the same time, rhBNP has the diuretic effect, when blood volume goes down PMSF can also decrease. The rhBNP decreases the VR resistance and increases the CO increased by reducing the stress volume in acute decompensated HF.The PMSF is the upstream pressure for the VR. It is linked to the circulating blood volume, as an important component of intravascular pressure.4.1The study had its own limitations includes the following:1.It was a retrospective study with a small sample size.2.The PMSF in this study was measured using the feasibility inspiratory hold method, a method that was minimally invasive monitoring at the patient's bedside and3.the limitations of venous return curve because it reflects the steady state data rather than the dynamic indicators, which needs clinical reasoning for interpretation. The study's strength was that there was no human study that had proved no significant differences between inspiratory hold method and circulatory arresting methods except in animal models.5This study concluded that rhBNP improves the CO both at 30 minutes and 3\u200ahours after administration in patients with acute decompensated HF. Through studying the the Guyton venous return curve, we could conclude that the role of rhBNP on CO was to reduce the peripheral circulation resistance at 30 minutes after rhBNP infusion and to reduce the resistance to VR at 3\u200ahours later.The technique of plotting Guyton venous return curve opens the door of our future studies. For example, Guyton venous return curve could be used as a tool to evaluate which kind of patients with decompensated HF could benefit from using rhBNP.Conceptualization: Xiaofei Zhang, Qiuye Kou.Data curation: Jianling Liu.Investigation: Zhi Liu.Methodology: Jiemin Li.Project administration: Zhenjie Wen.Resources: Ming Zhang.Writing \u2013 original draft: Jianling Liu, Xiaofei Zhang, Qinhan Lin.Writing \u2013 review & editing: Qiuye Kou."}
+{"text": "The role of definitive radiotherapy in advanced esophageal squamous cell carcinoma (ESCC), especially in the metastatic setting, remains unclear. Therefore, we aimed to investigate the efficacy of chemoradiotherapy (CRT) versus chemotherapy (CT) alone in these selected patients.We retrospectively evaluated 194 newly diagnosed advanced ESCC who underwent definitive CRT or CT alone, including 97 patients with locally advanced and 97 patients with distant metastatic disease. Cumulative overall survival (OS) and progression-free survival (PFS) were evaluated with a log-rank test. Propensity score matching was used to simulate random allocation. In addition, we performed subgroup analysis in the locally advanced and metastatic disease.p = 0.002) and PFS  in the CRT group were superior to that in the CT-alone group. Further subgroup analysis revealed that CRT conferred survival benefits to both locally advanced and metastatic cohorts. For patients with distant metastasis, median OS  and PFS  in the CRT group were superior to that in the CT-alone group. In a multivariate Cox regression analysis of the entire cohort, additional definitive radiotherapy was independently associated with better OS (p = 0.041) and PFS (p = 0.007).After matching, 63 well-paired patients were selected. The adjusted median OS (12.5 vs. 7.6 months, In both locally advanced and metastatic ESCC, additional definitive-dose radiotherapy was associated with improved clinical outcomes. Therefore, more consideration should be given to its application in the metastatic setting. Esophageal cancer (EC) is the seventh most common cancer worldwide and the sixth leading cause of cancer-related death, with approximately 572,000 patients diagnosed in 2018 \u20135. SinceWe retrospectively reviewed patients with newly diagnosed advanced ESCC who received CRT or CT alone at the Guangxi Medical University Cancer Hospital between June 2010 and May 2020. The institutional ethics committee approved this study, and informed consent was waived by the board. The eligibility criteria were as follows: (1) ESCC confirmed by histology; (2) clinically confirmed advanced disease (stage IVa or IVb) according to the 8th edition AJCC staging system ; (3) EasFor all patients, two- or three-drug cisplatin-based chemotherapy was administrated at 3-week intervals for up to 6 cycles as first-line therapy. For patients undergoing CRT, definitive-dose radiotherapy was administrated synchronously with 2 to 3 cycles of cisplatin-based chemotherapy. Radiotherapy was performed with intensity-modulated radiotherapy using a 6-MV linear accelerator  at five fractions per week. The gross tumor volume (GTV) and metastatic lymph nodes (GTVnd) were delineated with visible lesions based on contrast-enhanced simulation CT, PET, and endoscopic evaluation results. The clinical target volume (CTV) was defined as a 0.5-cm horizontal expansion from GTV/GTVnd, a 3\u20135-cm craniocaudal margin from GTV, and a 0.5-cm craniocaudal margin from GTVnd. The planning target volume (PTV) was determined by adding a 0.5-cm margin to the CTV. A median total dose of 60 Gy  with a median per dose of 2.0 Gy  in a median fraction of 30  was prescribed to the PGTV for five consecutive days in a given week. The dose constraints for organs at risk (OARs) were as follows: (1) lung: the whole lung V20 <28%, V30 <20%, and Dmean <15\u201317 Gy; (2) spinal cord: Dmax <45 Gy; and (3) heart: V40 <30% and Dmean <30 Gy.2 test. We performed multivariate Cox regression analysis to identify clinical variables independently associated with PFS and OS, and factors with p < 0.05 in the univariate Cox regression analysis were included. Statistical analysis was undertaken using R version 4.0.2 software, and p-values <0.05 were considered statistically significant.For posttreatment follow-up, enhanced CT and upper gastrointestinal endoscopy were reevaluated 1 month after treatment and every 3 months after that. Progression-free survival (PFS) was defined as the period between the date of initial treatment until disease progression or recurrence or death. Overall survival (OS) was defined as the period from initial therapy to censor or death. OS and PFS rates were evaluated using the Kaplan\u2013Meier method with the log-rank test. Continuous variables were compared with the Student\u2019s t-test, while categorical variables were compared with Fisher\u2019s exact or Pearson\u2019s \u03c7To minimize potential selection bias and confounders, propensity score matching (PSM) was used to control for differences in baseline characteristics. Using a caliper of width equal to 0.2 without replacement, patients in the entire cohort were matched at a 1:1 ratio to simulate random allocation. Covariates entered into the propensity model included body mass index, ECOG score, TNM stage, number of metastatic sites, absolute neutrophil count, and albumin level. All baseline covariates were balanced in the locally advanced disease and metastatic disease subgroups. Therefore, PSM was not performed in the subgroup analysis.2, p = 0.01), poorer physical performance , greater tumor burden , lower absolute neutrophil count , and lower albumin level . After matching, 63 well-paired patients were selected. There were no significant differences between the CRT group and the CT-alone group in baseline characteristics after PSM, as shown in A total of 194 patients with advanced ESCC were deemed eligible and assessed. Among them, 97 patients (50%) were locally advanced, and 97 patients (50%) had distant metastasis. The majority of patients with distant metastasis had a low systemic tumor burden. Seventy-seven (79.4%) patients had only one metastatic site , and 14 (14.4%) patients had two metastatic sites. Merely 6 (6.2%) patients had at least three or more metastatic sites. A total of 101 patients (52.1%) received CRT, and 93 patients (47.9%) received CT alone. The median cycles of chemotherapy for the entire cohort were 3 (1\u20136 cycles). Before propensity score matching, patients in the CT-alone group had significantly worse baseline characteristics compared to those in the CRT group, with a lower body mass index  patients died and 58 (29.9%) patients were right-censored. Before matching, the median OS and rates of OS at 6, 12, 24, and 60 months were superior in the CRT group to that in the CT group  and PFS . At the same time, the N stage and number of metastatic independently predicted only PFS .Univariable and multivariable Cox analyses for OS and PFS of the entire cohort before PSM are shown in Early toxicities that occurred in CRT and CT-alone cohorts were assessed according to the National Cancer Institute Common Toxicity Criteria for Adverse Events, version 4.0 (CTCAE 4.0) . The mosFourteen (22.2%) patients had grade \u22653 radiation esophagitis in the matched CRT group. One (1.6%) patient developed grade 3 radiation pneumonitis 4 months after RT. One (1.6%) patient developed grade 5 upper GI bleeding, and one (1.6%) patient developed grade 3 upper esophageal fistula, both at 3 months after RT. No patient experienced grade \u22653 radiation dermatitis. Twenty-three (36.5%) patients had grade \u22653 leukopenia, 21 (33.3%) had grade \u22653 neutropenia, 8 (12.7%) had grade \u22653 anemia, and 4 (6.4%) had grade \u22653 thrombocytopenia.In the matched CT-alone group, esophageal fistula occurred in 1 (1.6%) patient after one cycle of CT. Two patients (3.2%) developed grade 3 and 5 upper gastrointestinal bleeding after 2 and 3 cycles of CT, respectively. Four (6.3%) patients had grade \u22653 leukopenia, 6 (9.5%) had grade \u22653 neutropenia, 6 (9.5%) had grade \u22653 anemia, and no patient had grade \u22653 thrombocytopenia.p = 0.000), neutropenia (p = 0.000), and thrombocytopenia (p = 0.006).Patients receiving CRT had a significantly higher incidence of grade \u22653 leukopenia (p = 0.021) and median OS  than CT alone (The current study showed that combined definitive dose RT (\u226550.4) to the primary tumor with chemotherapy resulted in better OS and PFS than chemotherapy alone in advanced ESCC, even in the presence of metastatic disease, with manageable toxicities. In terms of metastatic EC, extended survival after definitive CRT has been reported by several previous studies. A prospective randomized phase 2 study demonstrated that the CRT was associated with significantly improved median PFS  before treatment was an independent prognostic factor for OS, further illustrating the importance of the nutritional state for patients with advanced EC. On the other hand, aggressive RT for primary tumor can reduce life-threatening events, including airway stenosis either by external compression or by direct tumor growth into the airways, fistula, perforation, and massive bleeding. It is reported that external beam RT could provide significantly more effective relief of pain and tumor-related complications for metastatic EC compared to esophageal stent placement  patients with metastatic disease had only one or two metastatic sites. We found no statistical difference in survival results between the locally advanced disease and metastatic disease. These results highlight that for advanced ESCC patients with low systemic tumor load, survival is most threatened by the failure of local control of the primary tumor. On the one hand, additional RT for primary tumor can effectively shrink the primary tumor and reduce dysphagia resulting from esophageal stricture. By increasing oral nutritional intake, RT may improve response rates, performance status, and long-term survival , 23. In lacement .p = 0.07) (p = 0.88) compared to RT alone, with increased toxicity  (p \u2264 0.001), while palliative dose (<50.4 Gy) CRT was associated with slightly inferior outcomes .Based on modern radiotherapy techniques, definitive RT \u226550.4) to the primary tumor may confer more significant survival benefits than palliative RT (\u226450.4 Gy) in patients with advanced EC. In the palliative setting, low-dose radiotherapy of less than 50.4 Gy is commonly used to relieve symptoms such as dysphagia, pain, and bleeding (0.4 to th = 0.07) . Another 0.0017) . Guttman 0.0017)  reportedp > 0.05) and regional control , but rather it seemed to increase the toxicity and higher treatment-related mortality rate. Notably, patients with squamous cell carcinoma account for the vast majority of the participants in this trial. Differently, radiation doses above 60 Gy are more frequently adopted in Asia. Several studies proposed that high-dose concurrent CRT of \u226560Gy could improve clinical\u00a0outcomes compared with standard dose (50\u201354 Gy) in EC (p < 0.000) compared with conventional-dose RT (50\u201354 Gy) in ESCC patients . The precise dose of definitive RT remains controversial. The landmark RTOG94-05 trial  failed t1, 96%. Ty) in EC ,\u00a028, esppatients . Howeverpatients . The optSafety findings in the current study were consistent with the known safety profile of CRT and CT alone \u201332. Signp = 0.001) and PFS  compared with chemotherapy alone as first-line treatment in patients with advanced ESCC /programmed death ligand-1 (PD-L1) therapies are currently the research hotspot and have demonstrated durable antitumor activity in patients with advanced EC \u201334. Accoced ESCC . As prevced ESCC , 36. Thiced ESCC . A phaseced ESCC .The present study had several limitations. Firstly, propensity score matching was used to reduce selection bias in this study. However, this led to the selection of patients and thereby decreased the sample size. Secondly, due to the retrospective nature of this study, data on quality of life were not available to us. Thirdly, we did not consider the changes in objective factors during the long-term period, such as increased applications of PET-CT, radiotherapy techniques, and chemotherapy regimens.In conclusion, additional definitive-dose radiotherapy was associated with improved clinical outcomes in locally advanced and metastatic ESCC. Therefore, more consideration should be given to its application in the metastatic setting.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Institutional Ethics Committee of Guangxi Medical University Cancer Hospital. The ethics committee waived the requirement of written informed consent for participation.Data curation: L-QL, Y-DW, and W-DZ, W-WM. Formal analysis: L-QL and Q-GF. Writing\u2014original draft: L-QL and Q-GF. Writing\u2014review and editing: T-SS. Funding acquisition: T-SS. All authors contributed to the article and approved the submitted version.This research was supported in part by the Guangxi Natural Science Foundation (CN) (2020GXNSFAA297171), China International Medical Foundation-Tumor Precise Radiotherapy Spark Program (2019-N-11-01), Guangxi Medical University Training Program for Distinguished Young Scholars, and Guangxi BaGui Scholars\u2019 Special Fund.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "In the wake of the international food crisis, many countries are paying more attention to food self-sufficiency to protect themselves from instability in the global food markets. Western Balkan region and the rest of the world are facing an array of challenges, including inflation and rising food prices. Recognizing the importance of producing sufficient food to cover a country\u2019s needs in circumstances of increasing risk of trade disruptions due to war or political tensions, this article aims to find the level of food self-sufficiency in the Western Balkan countries. The self-sufficiency ratio is calculated for different food groups and individual products over a 14-year period (2006\u20132019) based on the FAO data and calculation method. Also, using panel data, the impact of different factors\u2014arable land, rural population, fertilizers efficiency, temperature change, precipitation, and GDP per capita change\u2014on cereals self-sufficiency is estimated. Results showed that in the covered pre-crisis period, the Western Balkans achieved a satisfactory level of food self-sufficiency, suggesting that the region is ready to respond to future challenges. Four indicators positively and significantly affect cereal self-sufficiency: arable land, rural population, fertilizers efficiency and GDP per capita change, while one indicator\u2014temperature change\u2014had a negative and significant effect. This article can serve as a basis for post-crisis research on the topic. All five Western Balkan countries  have experienced a very turbulent history. For the former states of Yugoslavia, it included a closed economy, wars, and inflation in the 1990s, breaking up the state of Yugoslavia as well as institutional and economic reforms in the newly created states. Albania also had to implement several reforms, considering it was one of the most closed economies. Although more than two decades have passed since then, the results of the region\u2019s transition are not satisfactory. Its\u2019 progress toward becoming a functioning market economy is modest and uneven. Overall living standards decreased while the unemployment rate increased . For exaAs with the rest of the economy, the Western Balkans\u2019 agriculture still lags behind the European Union (EU) in terms of productivity ,7, althoWestern Balkan countries have a shared strategic objective\u2014accession to the EU. They are in the various stage of the accession\u2014the most advanced are Montenegro and Serbia, next North Macedonia and Albania, while for Bosnia and Herzegovina, on 12 October 2022, the European Commission recommended that candidate status be granted. However, despite the objective that unites the whole region and the EU, exposed to two types of pressures, often contradictory\u2014the EU\u2019s requirements and domestic interest groups\u2014Western Balkan countries rather choose domestic political economy interests than alignment with EU policy .If turning to one\u2019s own national interests at the expense of the EU was unacceptable in the past years, today, it is more understandable. Hunger is stalking\u2014the world is moving backward on eliminating hunger and malnutrition, away from the intended Sustainable Development Goals (SDGs). Food insecurity and malnutrition due to conflicts, climate extremes, economic shocks, and growing inequalities significantly increased. The number of people affected by hunger globally rose to as many as 828 million in 2021, an increase of 46 million since the COVID-19 pandemic started (2020); 350 million more were moderately or severely food insecure in 2021; a gender gap in food insecurity rose for more than 1% in 2021 compared to 2020 .2O5 + K2O production and imports in 2020, Serbia produces about 30%, and Bosnia and Herzegovina about 15% of its total fertilizers needs. The rest of the region\u2014Montenegro, Albania, and North Macedonia completely rely on imports. Dependence on imports in an open market that functions without interruption is not problematic. But in light of new events and the current crisis, all countries, including the region, dependent on imports will face various problems. The fertilizer supply shock is expected to cause severe consequences in the Western Balkan\u2019s food system. The farmers will either reduce their use, which will result in a decrease in yields or, like American farmers, they will begin to reduce the area under corn and wheat and increase the area under soybeans, which require less fertilizer [The conflict in Ukraine deepened the problem\u2014it has blocked ports; the price of freight and fertilizer costs increased due to rising fuel prices; social unrest and political violence are rising, adding to risk for the global economy; the number of internally displaced and refugees increased; many countries have empty reserves (due to spending during the pandemic); the already-climbing world food prices exploded due to de facto export blockade or the temporary bans. The dimensions of the consequences are understandable, knowing that Ukrainian and Russian wheat and barley account for almost a third of global exports, sunflower oil more than 70%, and Russia is the top global fertilizer producer . Moreovertilizer . As a cortilizer .Due to supply-side pressures, headline inflation in the region started picking up in the second half of 2021. As a result, the headline consumer price index (CPI) growth in August 2022 ranged from 16.8% to 8% across the region . In all countries, food price inflation contributed the most to headline inflation: the cost of food in August of 2022 increased over the same month in the previous year for 25.5% in Montenegro, 25.1% in North Macedonia, 24.68% in Bosnia and Herzegovina, 20.4% in Serbia, and 14.9% in Albania .main objective of this paper is to find the Western Balkan region\u2019s level of food self-sufficiency. Following the FAO recommendations, the additional objective of this paper is to analyze the inputs use effectiveness in the Western Balkans. Finally, this paper analyses the factors influencing the level of cereals self-sufficiency in this region.In this regard, the Although a few studies analyze food security in the Western Balkan region , they foOur sample is made up of five countries of the Western Balkan region ), over a 14-year period (2006\u20132019). The initial year of research is 2006 because it coincides with the gaining of independence of Montenegro. The end year (2019) is the most recent year available in the database. The selected time frame provides a good basis for exploring the region\u2019s potential to satisfy its population\u2019s food needs before the pandemic and the Ukrainian crisis and trade restrictions related to them . It is essential to report the situation before the crisis in order to monitor the post-crisis situation in future research.On the basis of the FAO calculation method , the fooThe countries with SSR < 100 produce less food than they consume. For example, if SSR is 25%, the country produces only 25% of the amount of food that it consumes. SSR = 100 means that the domestic food supply can satisfy domestic consumption; the country produces all amount of food that it consumes. On the other hand, the countries with SSR > 100 produce more food than they consume. For example, SSR = 140 means that 40% of the country\u2019s production can be exported or placed in Commodity Reserves. SSR was calculated for food groups  as well as for individual items: wheat, maize, barley, poultry, bovine, pig meat, mutton and goat, apples, grapes, tomatoes, onions, potatoes, beans, and sunflower oil, for each country, and the whole Western Balkan region. The same calculation method FAO use for the series of \u2018cereal import dependency ratio.\u2019 However, in the current crisis, which will potentially last for years, it is important to extend analyses to more commodities, not just cereals.2O5 + K2O), was used as a proxy for fertilizers.In the next step, we investigated the relationships between cereal production and population growth. After that, using data from the FAOSTAT database , we calcThe achievement of food self-sufficiency in a country depends largely on input use, so the impact of different factors on the self-sufficiency of cereals was analyzed. The independent variables which are included in the model are arable land per capita, fertilizers efficiency, rural population, annual mean temperature change, annual precipitation increase, and GDP (Gross Domestic Products) per capita PPP (Purchasing Power Parity) change.it represents SSR cereals in the Western Balkan country i in the period t; Fit represents fertilizers efficiency in the country i in the period t; Git represents GDP per capita PPP in the country i in the period t; Pit represents precipitation in the country i in the period t; Ait represents arable land per capita in the country i in the period t; Rit represents rural population in the country i in the period t; Tit represents temperature change in the country i in the period t; \u00b5i and \u03bbt represent cross-section and period-specific effects (random or fixed), respectively; and uit represents a random error of the model.All variables were transformed into natural logarithms. The basic form of the estimated model is:A). Variable temperature change (T) arose from several studies which showed that climate change could adversely affect the future availability of water and land, even in the case that their effects are milder than the effects of population change [R) and food self-sufficiency is most visible in the least developed countries that are all classified as net food importing countries [F) and annual precipitation increase (P) as variables. Usually, with economic growth, a country increases the ability to purchase food from abroad [Knowing that the countries that lack natural resources  are unable to achieve food self-sufficiency and depend on imports to provide food for their population , we use n change ,38,39. Iountries . Howeverountries . Productountries . That ism abroad , so we iJoint significance of differing group means, Breusch-Pagan, and Hausman test statistic.The selection of the appropriate panel model among the pooled Ordinary Least Square (OLS), Fixed-effect (FE), and Random-effect (RE) was based on the following tests: It is expected to find a positive relationship between the SSR of cereals and fertilizers efficiency . It has As shown in Calculation of the 5-year average food SSR showed that out of five Western Balkans countries, only one country (Serbia) had an overall SSR above 100% for most items . The othSerbia is self-sufficient in the following items: all cereals, including corn, wheat, and barley; some meat\u2014bovine and mutton and goat; milk; eggs; fruits total and some kind of fruits (apples); vegetables total and some individual vegetables item (onions); sugar crops; and sunflower oil. North Macedonia, unlike Serbia, is highly dependent on cereals and meat imports. It imports about 30% of its cereals needs, almost half of its wheat needs, and more than 70% of its meat. The most critical situation is with poultry\u2014an SSR is just about 7%. However, the country produces an abundance of fruits and vegetables and enough starchy roots to satisfy the demand of its population. Just like North Macedonia, Albania is dependent on cereals and meat imports. It imports about 60% of the wheat, 20% of corn, 40% of barley, 40% of poultry, and 40% of pig meat needs. Unlike North Macedonia and Serbia, it is not self-sufficient in fruits, even though it is close to reaching self-sufficiency in apples and grapes. The vegetable sector has better performance, and in addition to Serbia and North Macedonia, Albania has an overall SSR for vegetables  above 100%. The situation in Bosnia and Herzegovina is hard to explain\u2014Bosnia and Herzegovina consists of two Entities: the Republic of Srpska and the Federation of Bosnia and Herzegovina. For example, the Federation of Bosnia and Herzegovina is dependent on wheat imports, while the Republic of Srpska produces enough wheat to meet the demand. The whole country is self-sufficient only in mutton and goat meat and eggs. In other words, this country relies on imports for almost all foods. It imports more than half of wheat needs, above 10% of corn and 20% of barley, somewhat below 40% of meat total, more than 70% of pig meat and above 60% of bovine, nearly 90% of sugar and oil crops, almost 30% of fruits, 10% of vegetables, more than 30% of tomatoes, 25% of pulses, 10% of starchy roots, etc. The 5th ranked Montenegro is the most dependent country on food imports. It produces only sufficient quantities of mutton and goat meat to meet the demand. The country produces only 5% of cereals demand, 25% of meat, about 70% of milk and eggs, somewhat above 55% of fruits and vegetables, 72% of potatoes, etc. Although Montenegro has a significant coastline, it produces only 26% of its fish needs.A list of the Western Balkans\u2019 countries ranked by the self-sufficiency level is as follows: Serbia is ranked 1st, North Macedonia 2nd, Albania 3rd, Bosnia and Herzegovina 4th, and Montenegro 5th. Tendencies on SSR are shown in Thanks to Serbia, the region as a whole is well supplied with many agricultural products . The regThe region is characterized by long-term stagnation in livestock production. According to Statistical Office of the Republic of Serbia (SORS) data, in Serbia, livestock has been halved in the last 30 years. In the period 2006\u20132019, the number of pigs decreased from 3999 to 2868 thousand, and the number of cattle from 1106 to 860 thousand . In enhaThe current global population is 7.97 billion, and it is expected to be 9.7 billion in 2050 . Rates oIn the Western Balkans, during the observed period, the annual population decreased by an average annual growth rate (AAGR) of \u22120.005, while the rural population decreased by an AAGR of \u22120.013. In the same period, cereal production increases with 0.049 AAGR . Cereal Western Balkans use about two times less fertilizer  for its agricultural production than the EU ; imports almost four times less pesticides (14.5 kg/ha vs. 3.7 kg/ha in 2019); and has a lower share of land area equipped for irrigation (18.8% vs. 11.3%), but have just less than 10% lower cereals yield than the EU  Figure . AAGR fofertilizers efficiency has been continuously higher in the Western Balkans than in the EU. After 2009, fertilizers efficiency in the EU decreased by 19% for each ton of fertilizers produced, from 22.8 tons of cereal produced (per ton fertilizer) in 2019 to 18.5 tons of cereal (per ton fertilizer) in 2019. In the same period, fertilizers efficiency in the Western Balkans increased by 42.3%, from 19.5 to 27.8 tons of cereals produced per ton of fertilizer , there is a downward trend in economic access to food. Even more, the country has the second highest population living hunger in Europe (5.7% undernourishment rate) . Clapp , improvement in information sharing and data collection, development of the system of a rapid assessment of the food stocks, etc., are highly recommended.The COVID-19 pandemic started in 2020 and significantly influenced food security in the world. Additionally, the conflict in Ukraine has deepened this problem, as it has blocked ports, the prices of freight and fertilizer costs increased, the risk to the global economy is higher, the number of refugees increased, and many countries emptied their food reserves. In that context, the main idea of this paper was to estimate the Western Balkans\u2019 food security and answer the question of whether food shortage is coming to this region.This research showed that this region, as a whole, is well supplied with the majority of food products. This is thanks to Serbia, the only country with an overall SSR above 100% for the most analyzed items. So, the level of self-sufficiency varies across the Western Balkan countries, where Serbia is in the first position while Montenegro is the worst. Because of that, regional cooperation is crucial, and the Open Balkan initiative is one step forward to better cooperation, signed by Serbia, Albania, and North Macedonia. Although results showed that the region is well supplied with food products, it could be endangered in the crisis conditions. For example, bans on the export of certain types of basic foodstuffs from Serbia at the beginning of the Ukrainian crisis significantly disrupted the situation on the market. Therefore, in crisis conditions, certain situations can threaten the efficient supply of food to the market at any moment. This can be very pronounced in livestock production that has retrograde tendencies.Additionally, the results of this research showed a satisfactory level of input efficiency, most probably due to the extensive nature of agriculture in the Western Balkan region. This is very important for the continuation of sustainable food production, which will surely focus more and more on the efficiency of inputs in the coming period.Factors significantly influencing cereals\u2019 self-sufficiency in the Western Balkans are arable land, rural population, fertilizers efficiency, temperature change and GDP increase. In the context of crisis conditions, it is valuable information that fertilizers efficiency significantly contributes to cereals\u2019 self-sufficiency level, as the Western Balkans depend on the import of fertilizers. So, in this situation, all countries of this region need to strengthen domestic capacities in producing fertilizers to enable continuous food production.This research has limitations. Some factors, such as the role of trade policies, infrastructure, food prices, and agriculture\u2019s role in the overall economy that can affect food supplies and food self-sufficiency, have not been included in the statistical analysis. Thus, future research can be directed toward a more detailed analysis of factors influencing regional self-sufficiency. Also, further study is required to quantify the effect of Open Balkan (and/or similar initiatives) to discuss in detail the common regional market, pros, and cons. In order to achieve the highest possible self-sufficiency level, the investigation of the current diets, their modification, and the reduction of food loss and waste should be imperative. On the whole, future research can be directed toward a more detailed analysis of factors influencing self-sufficiency in the region."}
+{"text": "Adding to the behavioural science domain, the principal idea behind the study is to investigate the impact of an array of behavioural, psychological, and demographic factors on financial decision making. The study utilizes a structured questionnaire to collect the opinions of 634 investors using a blend of random and snowball sampling techniques. The partial least squares structural equation modelling has been used to test hypotheses. PLS Predict has been applied to estimate the out-of-sample predictive power of the proposed model. Finally, the multi-group analysis has been applied to assess the differences across gender. Our findings attest the relevance of digital financial literacy, financial capability, financial autonomy, and impulsivity on financial decision making. Additionally, financial capability partially mediates the nexus between digital financial literacy and financial decision making. Also, Impulsivity negatively moderates the relationship between financial capability and financial decision making. The overall results of this comprehensive and unique study portray the influence that various psychological, behavioural, and demographic factors have on financial decision making, favouring the design of a feasible and lucrative financial portfolio to ensure financial well-being of households in the long run. We investigate the research topic from a developing country perspective and consider India for the empirical investigation. This is because, India was one of the few countries which observed the longest and most stringent lockdowns in the world, with the GDP slumping by 24% causing major upheavals in unemployment rates (up by 24%) and massive slides in household income (down by 46%). Moreover, worsening macroeconomic indicators such as job losses, GDP deteriorations and accelerated inflations, have put working individuals in a financial dilemma, rendering more than 136 million workers financially vulnerable  during tFrom the perspective of extant literature, we offer an extension to the behavioural science domain. This is because, through generations, multidisciplinary schools of thought ,24 have The current void in literature and invalidity in prior findings serve as the main motivators for conducting a first-hand empirical study on personal, behavioural, and psychological factors affecting financial decision making. Also, given that financial capabilities and cognitive skills fluctuate with gender, further strengthening or weakening the impact of various determinants on FDM, this study investigates the influence of gender related differences on individual FDM.Altogether, we challenge the rational decision-making behaviour and propose the influence of positive and disempowering factors to achieve four-fold objectives in this study, i.e., firstly, to identify the personal, behavioural, and psychological factors affecting FDM. Secondly, to empirically investigate the association between various behavioural and psychological factors on FDM, aiming at extending the existing literature on FDM. Thirdly, to evaluate the moderating effects of gender and age on the determinants of FDM. Finally, to invoke implications from the results derived and offer recommendations for enhancing individual FDM. To accomplish these objectives, the study utilizes a structured questionnaire to survey the opinions of 634 household investors across the National Capital Territory (NCT) of Delhi in India to decipher the driving factors in FDM.A comprehensive literature review is done to identify the FDM factors and formulate eight testable hypotheses and the SmartPLS 3.3 is used for testing the hypotheses. The partial least squares (PLS) predict is applied to estimate the sample predictive power. The differences across gender are assessed using the multi-group analysis (MGA) technique. Findings reveal a positive association of DFL, FAUT, and IMP with FDM. Further, we observed a significant positive mediating role of FC in the DFL-FDM nexus.The remaining paper is structured as follows. Section 2 emphasizes on theoretical background, prior literature, and hypotheses formulation. Section 3 elucidates data and methodology, followed by discussion of results in Section 4. Section 5 concludes the paper with implications and outlines future research prospects.2FDM has harnessed significant attention in the past decade due to the incongruities evident in financial planning and diminished savings . It is bThat said, the unprecedented changes, complexities, and multidimensional facets of FDM compel us to complement the prospect theory through an earlier model, called decision making theory . The the2.1Extant research has deeply delved into the gender influence on FDM . The conH1Males and Females significantly differ in their financial decision making (FDM)More recently, Hsu et al. , conclud2.2The proliferation of digital financial services and platforms have aggravated the relevance of digital financial literacy (DFL) over financial literacy. Time has proven that financial literacy alone cannot help one to wade through the complexities in the digital world. Unfortunately, DFL is now an imperative for individuals to access financial products and services or make financial decisions. It refers to the knowledge, skill, and abilities to access digital financial services . The forThe goal framing theory posits that individuals endeavour to advance their goals which is over all wellbeing by engaging in various self-regulating behaviour. Furthermore, the theory assumes that human beings try to achieve conflicting/multiple goals, thereby self-motivating them to be engrossed in higher order cognitive functions. These multiple goals are then grouped as goal frames and decision-making behaviour is then ruled by any one or all the goal frames namely hedonic, gain and normative. While the hedonic goal frame focuses on the present feel good notion, the gain goal frame is related to judicious decision making which assures conserving resources and increasing income. The normative goal frame on the other hand refer to those heuristic behaviour that emerge from external factors. However, goal frames are not mutually exclusive, and the theory postulates the strength of a goal frame being confronted by the individual's current goals arising from unexpected situations . For exaH2Digital financial literacy (DFL) positively relates to financial decision making (FDM)Therefore, it should be noted that the goal frames extent of assertion in decision making is a cumulative effect of external factors as well the capacity to regulate one's behaviour. Here we stress that DFL will equip individuals with the confidence and skills to use financial platforms and services effortlessly, thereby removing the mental barriers that inhibit the usage and access towards such services. It will also enable users to intensify their digital financial management usage over simple internet usage  and take2.3Financial capability (FC) is a multi-dimensional concept that encompasses both judicious money management and rational decision making where individuals carefully weigh the expected costs with the benefits whilst being mindful of their financial constraints . HoweverH3Financial capability (FC) positively relates to financial decision making (FDM)H4Financial capability (FC) significantly mediates the relationship between digital financial literacy (DFL) and financial decision making (FDM)The concept has been underpinned by the capability approach, pioneered by Amartya Sen and Martha Nussbaum in 1980's who proposed FC as an outcome of the external socio-cultural environment one is exposed to apart from being the conversion factor which transforms available resources  into valuable resources (pension) . Prior r2.4The current paper explores financial attitude (FA) as a psychological factor impacting financial decision in three forms, namely decision to consume, decision to save or decision to invest . FA is aH5Financial attitude (FA) has a positive relation with financial decision making (FDM)Furnham  outlines2.5Impulsivity, according to Barberis and Thaler , is a psDickman and Meyer  argued tAs an attempt to bridge the aforesaid gap, we argue that IMP  drives an individual to unplanned, hasty, and suboptimal financial decisions to reap short term benefits without looking at the potential negative long-term impact of the decision. IMP leads to risky financial decisions such as over-spending and over indebtedness  as emotiH6Impulsivity (IMP) negatively relates to financial decision making (FDM)H7Impulsivity (IMP) significantly moderates the relationship of financial capability (FC) with financial decision making (FDM)Early studies have already factored in this variable  in behav2.6Financial autonomy (FAUT) refers to the limited dependency on others albeit having the capability and liberty to engage in FDM. As a research topic, the nexus of FAUT as a driver of FDM has remained under-explored . VosylisJariwala and Dziegielewski  add thatH8Financial autonomy (FAUT) positively relates to financial decision making (FDM)That said, the research in FAUT-FDM nexus is limited, as extent research has either unwrapped the determinants of FAUT  or explo33.1To investigate the determinants of FDM, we conducted a questionnaire-based survey. The empirical data was collected from the respondents located in the National Capital Territory (NCT) of Delhi, India using the mix of random and snowball sampling. Since this study requires a respondent to be an investor, thus a mandatory check question was added, i.e., whether the respondent invests in any kind of financial instrument and accordingly respondents were allowed to proceed further only when the answer was \u2018yes\u2019. The Likert 5-point measurement scales were used to test the theory, in alignment with the previous research (see Annexure 1). The questionnaire was managed using google forms which elaborated the objective of the study in the introduction section. The responses were collected between February 2022 and August 2022.The survey clearly mentioned about the voluntary participation of the respondents (due consent was taken from the respondents before proceeding with the survey) and assured that their responses would be treated with utmost confidentially whilst maintaining anonymity. The willingness to complete the questionnaire is an expression of respondent's consent to take part in the research. It was compulsory to attempt all questions that were asked in the google form, eliminating any chances of having missing data. The questionnaire consisted of three sections i.e., independent variables  in section one; mediator (FC) and dependent variable (FDM) in section two; demographic profile in section three. The study focused on Delhi as it was the worst Covid hit area with the exponential rise in cases leaving the city far behind the financial capital Mumbai which was once the largest COVID hotspot in the country. This led to further lockdowns, thus crippling the economy, and leading to catastrophic repercussions on financial decision making and wellbeing.The sample size sufficiency was ensured by the G*Power software via a priori analysis . With a 3.2Financial autonomy (FAUT) was conceptualized as the higher order construct comprising of three lower order reflective constructs or dimensions namely emotional, functional, and reflexive. The questionnaire and scales used in the present study has been adopted from previous studies on the topic of research, as presented in The scales have been measured on five-point Likert scale, 1 representing strongly agree and 5 strongly disagree. Annexure 1 shows the items with respect to each of the construct. All these scales have high validity and reliability, as shown in 3.33.4To assess the common method bias, the bivariate correlations between the constructs has been checked and all values were found to be less than 0.90 . As a seThe SmartPLS 3.3  has been4SmartPLS 3.3 software  has been4.1As presented in The constructs exhibited satisfactory discriminant validity see  with all4.2As per the existing literature studies, FDM is influenced by gender. Thus, to analyse this, we have conducted the multi-group analysis (MGA) based on the gender of a respondent. To conduct MGA, the measurement invariance must be established using the MICOM procedure ,88. The ns) and FC (\u03b2\u00a0=\u00a00.657* \u2192 0.590*) which is strong and significant in case of males. Although FAUT relation becomes insignificant in case of females (\u03b2\u00a0=\u00a00.167ns), IMP has been negatively related to FDM in both the groups, with more significance in case of females (\u03b2\u00a0=\u00a0\u22120.155* \u2192 \u22120.106*). However, moderating effect of IMP on the FC and FDM relationship becomes insignificant across both the groups, with similar results being applicable to FA as well. Thus, the full sample results have been considered for the study.The p values in the last column of 4.3The bootstrapping technique was conducted to evaluate the hypothesis with 10,000 subsamples, the corresponding 95% bias-corrected confidence interval . In the After assessing the overall structural model by way of bootstrapping, the researchers have also assessed the predictive ability of the proposed model. The out sample predictive power has been estimated using the PLS Predict with 10 folds and 10 repetitions. 5The current study contemplates behavioural, psychological, and demographic factors impacting financial decision making (FDM) as an outcome of the interaction among individual behavioural differences, life experiences, practices, and formal instruction with each one developing content knowledge, attitude, competencies and skills towards financial topics and financial domain. The first hypothesis proposed varied trends in FDM in males and females, but results report a contradiction to the former, thus signalling gender irrelevance in FDM . This caIn continuum, hypothesis 2 assumes a direct relationship in the DFM-FDM nexus with results emerging statistically significant to attest the former proposition. We express our congruence to the goal framing theory and findings of Azeez and Akhtar , where tThe next set of hypotheses  and H4 iEmbarking on other determinants of FDM, The effects of IMP on FDM as well as the moderating effects of IMP in the FC-FDM relationship were the next focal points of attention in Lastly, 6This study reiterates the importance of adopting an integrative approach for better decision making where each system can derive optimal results under varying situations, hence corroborates the recommendation provided by Hochman et al. . This isIn our literature review, we came across various schools of thought and formulated eight hypotheses for testing the nexus of FDM with a list of psychological and behavioural factors, such as positive FAUT, FA including IMP and materialistic attitude, sound DFL and FC as cumulative effects of experience, skills, and attitude, as well as mediating roles of selective factors in the nexus. Moreover, as a major area of focus, we investigated the gender influence on FDM, and observed limited and inconclusive take of researchers on IMP and FA and hence examined varied trends in FDM from the gender perspectives but found no significant FDM behavioural difference between males and females.Based on our empirical findings, we can claim making some contributions to the extant literature on FDM. For example, we observed a positive association of DFL, FAUT, and IMP with FDM. Further, we observed a significant positive mediating role of FC in the DFL-FDM nexus. Based on our literature review, we however failed to trace any research that has explored any FC-FDM nexus or a mediating influence of FC in the DFL-FDM nexus. Given that DFL is a nascent concept, hence very few papers have surfaced in this domain and none till date has attempted to make any empirical investigation on the direct nexus of DFL with FDM. We claim the incorporation of DFL in this study and evidence of its influence on FDM, are a contribution to the existing literature. Moreover, this study investigated the direct impact of IMP  on FDM and its moderating effect in the FC-FDM nexus.We argued that IMP overpowers one's FC and drives an individual to unplanned, hasty, and suboptimal financial decisions to reap short term benefits, without looking at the potential negative long-term impact of the decision (FDM). We spotted a negative moderation effect of IMP in the FC-FDM relationship and hence claim another contribution to the extant literature. Further, we investigated varied trends in FDM in males and females but found no significant difference between them in FDM. Given that the direct effect of DFL on FDM is more in case of males as compared to females, we emphasise creating more DFL opportunities for women. Moreover, as a pioneering study to decipher a new empirical model to comprehend the FDM process, we claim to fill some voids in the extant literature given that prior studies ,95 only 6.1Although the sample and the research design adopted in this study are adequate to achieve the desired research objectives, like any other research, this study is also susceptible to few limitations. The sample size sufficiency was ensured by the G*Power software in this study. However, given that provinces/states in India are highly diverse in nature, we believe that use of a larger size of sample with respondents from various states could provide a more robust picture of the FDM behaviour of an average citizen in India and enable greater generalisability of the findings in the contexts of the developing world with similar socio-economic features. Also, given that \u201ccountries with higher rates of savings have had a faster economic growth than those with lower saving rates\u201d , furtherThis study provides an empirical and conceptual springboard for ensuing work on other potentially important determinants of FDM behaviour. Given that behavioural finance is a relatively new but a fast growing field where the list of psychological factors will continue to expand and new opportunities and challenges arise, further studies can be planned by considering other psychological factors such as regret bias, gender IMP, anchoring effect, defence mechanisms, personality traits , over and under reaction, mental accounting, and so on to examine their likely impacts on investors\u2019 FDM attitudes. As successful research outcomes, many of these factors might turn out to be useful determinants of the risk-taking and FDM behaviour of the household investors.Also, in the wake of ongoing automations and digital transformations of financial services, we emphasise further studies on exploring relevant behavioural factors such as prejudice and general mindset to DFL, emotional intelligence, among others and their influence on FDM, with particular focus on gender bias. Also, in consideration of the fact that the global digital skills crisis is worsening due to growing automation and use of artificial intelligence , and digIn order to develop further insights into this topic and minimise the rising knowledge gap in literature with regard to the nexus between gender and FDM , it woul6.2The findings in this research would enable financial regulators and policymakers to better understand the nature and type of the influence that psychological and behavioural factors have on household investors\u2019 FDM. In the ongoing post-pandemic recovery period, this research will be beneficial for financial professionals, regulatory bodies, or investment advisors in understanding the behavioural patterns in FDM that are likely to be associated with the economic and financial market volatilities. This study also will help investors to be aware of the impact of their own psychological factors on their FDM and accordingly enhance the rationality and efficiency of their investment decisions. Also, given that we have attempted to minimise the differential gap between India and similar developing economies from a demographic perspective by investigating the gender-FDM nexus in the context of India, we emphasise that further exploration about this relationship in connection with the listed behavioural and psychological factors will enable household investors to understand their market behaviour better and make candid decisions regarding their investments from inheritances and/or family savings.Rekha Pillai: Analyzed and interpreted the data; Wrote the paper.Parul Kumar: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.Md Aminul Islam: Contributed reagents, materials, analysis tools or data.Taimur Sharif: Contributed reagents, materials, analysis tools or data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Data will be made available on request.The authors declare no conflict of interest."}
+{"text": "Automated blood-vessel extraction is essential in diagnosing Diabetic Retinopathy (DR) and other eye-related diseases. However, the traditional methods for extracting blood vessels tend to provide low accuracy when dealing with difficult situations, such as extracting both micro and large blood vessels simultaneously with low-intensity images and blood vessels with DR. This paper proposes a complete preprocessing method to enhance original retinal images before transferring the enhanced images to a novel blood-vessel extraction method by a combined three extraction stages. The first stage focuses on the fast extraction of retinal blood vessels using Weighted Kernel Fuzzy C-Means (WKFCM) Clustering to draw the vessel feature from the retinal background. The second stage focuses on the accuracy of full-size images to achieve regional vessel feature recognition of large and micro blood vessels and to minimize false extraction. This stage implements the mathematical dilation operator from a trained model called Dilation-Based Function (DBF). Finally, an optimal parameter threshold is empirically determined in the third stage to remove non-vessel features in the binary image and improve the overall vessel extraction results. According to evaluations of the method via the datasets DRIVE, STARE, and DiaretDB0, the proposed WKFCM-DBF method achieved sensitivities, specificities, and accuracy performances of 98.12%, 98.20%, and 98.16%, 98.42%, 98.80%, and 98.51%, and 98.89%, 98.10%, and 98.09%, respectively. Initially, human experts primarily used digital retinal images in ophthalmic clinics to identify DR and related eye diseases. The retinal blood vessels are important diagnostic indicators for DR and the pathologies of systems within the human eye. More and more research indicate that the accurate extraction of retinal blood vessels is helpful in the analysis of other related ophthalmic diseases. The manual extraction of blood vessels requires an expert ophthalmologist\u2019s skills. Although such manual extraction is possible, it is time-consuming and there can be human error when working with large image datasets. Therefore, automated systems for the precise extraction of retinal blood vessels are urgently needed to reduce the workload of expert ophthalmologists. An example of the automatic extraction of blood vessels is illustrated in Retinal images often require higher contrast and quality, making it difficult to extraction the Region of Interest (ROI).Most retinal images suffer from imbalanced illumination and noise , making it difficult to distinguish the blood vessel from the background image.Retinal blood vessels come in many shapes, sizes, and unexpected forms, making it challenging to identify large and small blood vessels.Considering the typical retinal blood vessels and the background region information in the digital retinal images shown in In these circumstances, research on vessel extraction from retinal images has attracted a large number of researchers.Related works and state-of-the-art studies enlighten this paper\u2019s proposed method, which is based on machine learning and deep-learning methods. A selection of studies on retinal vessel extraction from 2015 to 2022 was reviewed to ensure that the summary of extraction techniques is as up-to-date. In choosing the articles, keywords such as blood vessel classification, blood vessel extraction, retinal imaging, DR segmentation, exudate segmentation, and red lesion extraction were selected. I searched for articles from websites such as Web of Science, PubMed, Science Direct, and IEEE Xplore. This review includes 216 papers from Q1 to Q4 journals and international conferences. Image extraction generally involves classifying ROI using specific image intensities or geometric features to suppress the unwanted background. This way, only specific areas are extracted as retinal blood-vessel regions. Typically, the anatomy of the retina consists of observable characteristics, such as the optic disc, retinal blood vessels, and eye pathologies that appear in retinal images. In addition, the researchers used retinal images from various data sources in their analysis, captured at different times, resolutions, and intensities. Accordingly, determining whether retinal blood-vessel extraction achieves the same accuracy as that of an expert diagnosis is a challenging task that has attracted many researchers.Recent implementations of deep-learning algorithms for retinal-image extraction were proposed by Soomro et al. . Imran eFor image processing, deep learning techniques, particularly Convolutional Neural Networks (CNNs), have received much attention ,21. DeepJiang et al.  suggesteIn addition to proposing a fully convolutional network, Atli and Gedik  were theMishra et al.  suggesteDeformable convolution was suggested by Jin et al.  for clasIn order to extract micro blood vessels, Dharmawan et al.  presenteThe region of interest has been identified and feature representations have been strengthened in blood-vessel extraction via the attention mechanism . Luo et There is a need for training samples with clear labels. Even though there are many retinal images, collecting annotated data is quite challenging because it involves qualified experts, takes a lot of time, and is expensive.The current retinal image samples are of low quality. As a result, deep learning models cannot develop more robust feature representations. The performance of the proposed methods is hampered by image noise, low intensity, and various features of DR diseases.There is an issue concerning training sample class imbalance. The performance of networks is harmed by disparity in the amount of positive and negative training examples. Class imbalance affects large and micro blood vessels, DR, and backgrounds. Because there are more non-blood-vessel pixels than blood-vessel pixels, deep learning models frequently categorize pixels in boundaries as non-blood-vessel pixels.Since the error extraction of micro blood-vessel pixels has less impact on the overall loss, the network performs poorly on micro blood vessels than on large blood vessels.When using deep learning to extract retinal blood vessels, the following issues have been reported:This research focuses on retinal blood-vessel extraction, with an emphasis on an extraction method that does not require specialized hardware for training the developed methods, decreases computational time, eliminates hyper-parameter adjustment, and minimizes memory. I chose the WKFCM-DBF method because of its many advantages. WKFCM-DBF identifies retinal blood vessels without a ground-truth image. This is a great advantage, as large datasets are typically unavailable for large-scale screening programs. In addition, Dilation-Based Functions and optimal thresholding are based on attributes of micro blood vessels in retinal images.The remaining portions of the article are divided into the following sections. A detailed description of the data preparation, image normalization, color space selection, noise removal, and Optic Disc (OD) feature extraction is provided in This section details a proposed method for extracting retinal vessels. The original image contrast was enhanced using preprocessing techniques, such as separating the RGB image to the green channel component, normalizing the colors with averaging threshold optimization, and applying the median filter on the improved image to decrease noise.A morphological Dilation-Based Function was applied to maximize retinal blood-vessel detail. Then, I used optimal global thresholding to improve binary image quality.Using images enhanced for accurate extraction allows well-developed algorithms to extract more retinal blood-vessel details. Because they have more shapes, positions, and sizes, I was able to extract them more effectively. Additionally, micro blood-vessel connections support the reuse of low-quality images in unseen images. This is important for extraction. The accuracy of specific proposed methods was also very high. Augmented images based on morphological Dilation-Based Function are a viable alternative to improve the performance of micro blood-vessel extraction and collect more spatial information while preserving the running time of the extraction process. I selected suitable clusters of WKFCM parameters to acquire images of large and micro blood vessels.Using a suitable technique, I was able to pay more attention to retinal blood vessels, especially micro blood vessels, if the correct parameters were used. I used enhanced images and a dilation function to address imbalance problems, resulting in binary images via different parameters. The optimal parameter thresholds  were empirically examined and applied to remove non-blood-vessel components. With this approach, the shape of retinal blood vessels can be better visualized to help expert ophthalmologists diagnose disease.The proposed WKFCM-DBF method was evaluated using three different datasets after training samples from each dataset. I was able to carry out cross-validation for additional validation. I tested the proposed method on the unseen or private dataset. The preprocessing steps, including color normalization with averaging threshold optimization and application of the median filter to remove noise in enhanced images to enhance image quality, are detailed below.I separated green, red, and blue channels from the RGB retinal image and then selected the green image. I identified the following crucial factors as being essential for accurate and reliable retinal blood-vessel extraction:Image enhancement is an essential preprocessing step for improving details in the retinal images. A simple averaging threshold optimization technique enhances image contrast and maintains the average brightness . The hisaverage, enhanced is the average brightness of the enhanced image \u00ceenhanced. The variable H represents the entropy of the maximized value of the information provided by Equation (3):The image threshold is calculated via Equation (1).enhanced is obtained using the averaging threshold optimization technique, with brightness improved by 0.58 as illustrated in The objective function The second preprocessing stage is the selection of suitable color channels for the extraction of retinal blood vessels. As shown in The object of retinal blood-vessel extraction is to segment large and micro vessels as much as possible. Based on the work of image preprocessing, the first step in this section was to apply the standard FCM method to extract retinal blood vessels. After that, I found that the optimal function for each extraction stage became the subsequent critical optimization. The optimization function of blood-vessel extraction based on the WKFCM-DBF method was investigated in that part. To train and test images for the extraction stage, preprocessed images, as described in 1, x2, \u2026, xn} be the number of examples provided. Then, c fuzzy clusters are provided with a probabilistic cluster partition of X by minimizing the within-cluster sums of the squared error objective function. The image extraction algorithms based on the FCM analysis are fuzzy optimization algorithms [m represents the sum of squared error criteria, its minimization produces fuzzy clusters by the membership matrix U, V is the set of cluster centers, C represents the total number of clusters, N denotes the total number of objects in any gray image in the dataset, dik represents the distance measure between object k and cluster center i, and uik represents the degree of membership of kth pixel belong to the ith cluster that satisfies the condition k  represents the kth pixels vector of pixels of a gray level image, vi {vi = 1, 2, \u2026, C} represents the ith cluster for i = 1, 2, \u2026, N, uik is the fuzzy membership degree of pixel xi in cluster k, m represents the index of the fuzzy weight , ||\u2026|| represent some inner product induced norm, and ||xk \u2212 vi||2 is the squared distance between the data element xk and the cluster center vi  [ik) and the new cluster centers (vi) are defined as Equation (7).FCM achieves clustering by repeatedly searching for a set of fuzzy clusters and their corresponding cluster centers that represent the structure of the data. The clustering algorithm depends on the user to indicate the number of clusters present in a dataset to be clustered. Let c be the number of fuzzy clusters (1 < c < n) and X = {xgorithms ,49. Thisgorithms .(5)Jm . The valHowever, the traditional FCM is essentially a local random search clustering algorithm based on Gradient Descent (GD) and thus has a greater dependence on the initial conditions.The WKFCM method has been derived from the traditional FCM based on modifying the kernel function, the membership degrees, and the fuzzy cluster optimization described in the following discussion ,54,55. Ttion (9) .(9)Kx,yThe objective function of Equation (11) can be expressed by Equation (12).i and the new fuzzy membership-degree value uik are calculated using Equations (13) and (14).Then, the cluster centroids vThe data points were normalized so that a hypercube bounds them. Given the data set Z, I set the number of clusters (c) at 2, the weighting exponent (m) in the FCM at 3, and the tolerance of termination (T) at 120.The clusters of centroids were computed by the standard FCM method.The clusters of centroids (step 2) as were regarded as input to the WKFCM.The new membership values of the data points were computed by the WKFCM method, using Equation (14).The value of \u2206 was calculated, using Equation (15):The clusters of centroids were updated by WKFCM, using Equation (13).The new membership values were updated by WKFCM, using Equation (14).The remaining steps and calculations of the WKFCM are the same as those of the standard FCM. I formalized the WKFCM algorithm as follows:I applied the preprocessing method to all datasets, which had been normalized and enhanced for the coarse extraction stage. Then, I used the WKFCM for 9  with different random initialization processes of the membership values of an object to all classes in the dataset. For random initialization for the kernel function, I used three kernel function values  of the adjustable parameter, \u03c3, of the Gaussian kernel function to find the optimized result out of the three. Next, I provided the optimized values of the number of clusters for five different validity measures  for the best choice among five values of the WKFCM obtained in 150, the maximum number of iterations. Finally, an initial threshold value (T) of 120 was selected as the minimum value to classify the histograms between the two classes. The blood vessel extracted using the proposed WKFCM method is illustrated in While extracting blood-vessel structure, a large and micro-vessel extraction will also run simultaneously with the same input image. However, the input image is converted to binary pixels to extract blood vessels from background pixels. As mentioned, the phase will be the retinal blood vessel in white pixels and the background with black pixels. During the coarse extraction, I found the loss of micro-vessel pixels. Therefore, I preserved the micro vessel while maintaining a computation time and superimposing their results in the original images. The connectivity between the current pixel and every one of its surrounding neighbors was examined using a morphological dilation operator. If connected, I categorized them into the same group. Accordingly, I adopted the morphological dilation operator in this stage, instead of the WKFCM false extraction, using Equation (16).The final stage of blood-vessel extraction is post-processing, in which images overlap between the extracted and the ground-truth image. Here, overlap means combining the extracted and the ground-truth images into one by simply applying the mapping coefficient to both. In this stage, the Mapping Coefficient (MAPC) is a statistical indicator that can determine the similarity between ground-truth and extracted images. MAPC is calculated via Equation (18).The following subsections provide a performance analysis by comparing the proposed preprocessing techniques, a novel WKFCM-DBF method, and state-of-the-art algorithms. To validate the effectiveness of the proposed method for extracting blood vessels, I compared my approach with those of previous studies.DRIVE: A collection of retinal images from the Netherlands, covering a wide age range of patients .STARE: A collection of 80 retinal images from the United States .DiaretDB0 : 130 retinal images from Kuopio University Hospital .Many publicly available retinal datasets detail retinal anatomy and blood vessels. This is essential in retinal analysis and blood-vessel extraction for training and testing algorithms on retinal databases. I evaluated the extraction method using three publicly available datasets:I used the retinal image of the DRIVE dataset from a DR screening program in the Netherlands. There were 400 diabetic patients in the screening program, ranging in years of age from 25 to 90. Forty retinal images were randomly selected from 400 diabetic patients, of which 7 images showed symptoms of DR and the remaining 33 images did not show any signs of DR. Each image was captured in JPEG format using a Canon CR5 non-mydriatic camera with a field of view of 45 degrees, 8 bits per color channel, and 768 \u00d7 584 pixels. There was a total of 40 images in DRIVE\u2019s training and test sets. This database has two manual classifications; I used 12 images as training ground truth and the remaining 28 images to compare the proposed extraction method with manual extraction by an experienced ophthalmologist. STARE was conceived in 1975 by Michael Goldbaum, M.D., of the University of California, San Diego. The STARE dataset has 80 images corresponding to the ground-truth images used for blood-vessel extraction, 49 of which are normal images, while the remaining 31 images show various types of abnormal disease. The first expert manually marked retinal images in large blood vessels, while the second and third experts marked micro blood vessels. Extraction results are commonly used as a ground-truth image for computing performance. Each image was captured in PPM format using a TOPCON TRV-50 camera with a field of view of 35 degrees, 605 \u00d7 700 pixels, and 8 bits per color channel. Finally, I obtained the retinal image of the DiaretDB0 from Kuopio University Hospital. The screening population consisted of 130 color retinal images, of which 110 showed symptoms of DR and the remaining 20 did not show any DR. Each image was captured in JPEG format with a field of view of 50 degrees, 1500 \u00d7 1152 pixels, and 8 bits per color channel. The information from the three selected datasets is summarized in TP represents vessels correctly extracted by the proposed method.FN represents retinal vessels extracted as non-vessels by the proposed method.TN represents non-vessels correctly extracted by the proposed method.FP represents non-vessels incorrectly extracted as vessels by the proposed method.To describe the performance of the blood-vessel extraction method, more than an accuracy performance is needed to determine the quality of extraction methods. In this section, I applied mathematical metrics that are commonly used to measure the performance of blood-vessel extraction algorithms: sensitivity, specificity, and accuracy. The extracted binary vessel image was compared pixel-to-pixel with its corresponding ground-truth image from the test set to calculate the extraction algorithm with sensitivity, specificity, and accuracy. The retinal vessel that was extracted was converted to a binary image to distinguish the vessels from the retinal background. A ground-truth image, manually marked by an experienced ophthalmologist, was used to evaluate the performance of the vessel-extraction method. Four parameters\u2014TP (True Positive), TN (True Negative), FP , and FN \u2014indicated that proposed method correctly and incorrectly extracted the blood vessel image with the ground-truth image marked by expert ophthalmologists.In this theory, sensitivity, specificity, and accuracy can be calculated via Equations (19)\u2013(21).Sensitivity (SEN) represents the correctly extracted blood vessels as blood-vessel pixels by the proposed method. Specificity (SPEC) represents the accurately extracted non-blood-vessel as non-vessel pixels by the proposed method. At the same time, accuracy (ACC) represents the proportion of correctly extracted pixels as blood-vessel or non-blood-vessel pixels.The proposed method was simulated within a MATLAB environment  and executed on a personal computer with an Intel (R) Core (TM) i7-6700K CPU at 4.00 GHz and 8 GB DDR3 RAM. To confirm the proposed method in retinal vessel extraction, I visualized and compared the activations from the different stages presented. If the proposed WKFACM-DBF method is used for non-preprocessed images or images with noise or artifacts, the value of SEN and SPEC may be decreased.If the proposed WKFACM-DBF method is applied to preprocessed images or noise-free images, the application of the proposed method results in higher accuracy than that of the non-preprocessing alternative. However, these results show that the proposed method performs best in the studied retinal blood-vessel extraction method, even when the noise level is high. However, as it is more effective on unseen images and the different datasets free of noise, the proposed method performs as well as the training dataset.As previously mentioned, the proposed method has been developed based on the data from a publicly available dataset. Therefore, even though this is a machine learning implementation, the extraction method is still biased for the different datasets. The proposed method with preprocessing performs better on the STARE dataset than on the DRIVE and DiaretDB0 datasets. The results for the DiaretDB0 dataset indicate that its accuracy was the lowest among all datasets, implying that its ability to correctly classify retinal blood vessels is not as good as that of the other datasets. Nevertheless, the accuracy of the DiaretDB0 dataset is still high (98.09%). Below is an illustration of a detailed analysis of the numerical results presented in Next, the proposed WKFACM-DBF method was compared to the traditional FCM method and tested on the DRIVE, STARE, and DiaretDB0 databases. From The proposed WKFACM-DBF method consistently outperforms standard FCM algorithms on these three measures. Note: the significant improvement in ACC scores over scores with standard FCM indicates that the proposed algorithm can better segment more micro blood vessels. This demonstrates, in particular, that the proposed improvement method highlights blood vessels of various widths.The kernel functions technique has a good extraction ability and has been improved into the fuzzy function in the WKFACM-DBF method. The optimal number of clusters and fuzzy weighting can improve the clustering accuracy. Therefore, the WKFACM-DBF method performs better than the traditional FCM algorithm in extracting retinal blood vessels with similar structures.The following is an illustration of a detailed analysis of the numerical results presented in The performance of the WKFACM-DBF method on the private dataset was analyzed. The private dataset contains 1800 retinal images (750 \u00d7 750 pixels) with a 45-degree field of view. Of all the retinal images, 200 contained DR lesions. The training set (30%) and the test set (70%) were divided. Each image in the training set had only one manual annotation, while the test set had three manual annotations provided by three experts. I used the same evaluation methodology as that of the publicly available datasets and used the ground-truth image annotated by three experts for performance evaluation. I compared the performance of the WKFACM-DBF method using the same parameters. In the private dataset experiment, the proposed method obtained 95.38%, 95.60%, and 95.42% for SEN, SPEC, and ACC, respectively. The proposed method achieved slightly lower SEN, SPEC, and ACC. However, incorporating the preprocessing approach and the WKFACM-DBF method with an optimal parameter improved the technique\u2019s overall performance.The performance comparison with other algorithms was carried out via a single-dataset test (the DRIVE dataset). The proposed WKFACM-DBF method does not have a training process and does not necessarily have single or cross-dataset tests. However, to fairly compare the performance of the proposed WKFACM-DBF method with other methods, I compared the results from the proposed method with the results from the same dataset test of the other methods. Each method\u2019s best SEN, SPEC, and ACC values are highlighted in bold in The proposed WKFCM-DBF method outperforms state-of-the-art methods based on pixel-based evaluation metrics, including SEN, SPEC, and ACC (most of the time) on three published datasets, DRIVE, STARE, and DiaretDB0, respectively. A careful preprocessing step of the proposed method\u2019s activation helped prove the hypothesis and increase the proposed method\u2019s efficiency by preserving retinal quality information in the extraction stage. This critical hypothesis was validated based on the SEN, SPEC, and ACC metrics. Moreover, the average time required to process one image on a computer with 4.00 GHz Intel(R) Core (TM) i7\u20136700K CPU, 8GB (RAM) under the Microsoft Windows 10 32\u2013bit operating system for all datasets was 4 s per image. The similar execution times for all datasets were due to the normalized and resized sizes of the original images before a feed to the extraction method. As a result, the WKFCM-DBF method was computationally efficient and fast compared to many state-of-the-art techniques. This study focused on developing a novel method that applies to blood-vessel extraction contaminated with noise or artifacts. I used a new hybrid clustering method for retinal vessel extraction. This paper\u2019s contribution is mainly related to three steps. I first described a normalization based on the averaging threshold optimization technique by conducting a rigorous analysis of the proposed method. Next, I described a noise-reduction algorithm based on a median filter method. Then, the images from the preprocessing stage were improved, contributing to a more reliable extraction of blood vessels and consequently improving the method\u2019s overall performance. I developed the retinal blood-vessel extraction method based on the traditional FCM method and its improvements. The developed method focuses on the fast extraction of blood vessels using Weighted Kernel Fuzzy C-Means (WKFCM) Clustering to draw the vessel feature from the retinal background. I enhanced the membership degree and the kernel employed in the objective function of the traditional FCM clustering approach to address its disadvantages. Then, the method focused on the accuracy of full-size images for regional vessel feature recognition of large and micro blood vessels to minimize false extraction. This method implemented the mathematical dilation operator from a trained model called Dilation-Based Function (DBF). Finally, an optimal parameter threshold was empirically determined to remove non-vessel components in the binary image and improve the overall blood-vessel extraction results.The new WKFCM-DBF method was thoroughly evaluated on both actual normal and abnormal images, and based on SEN, SPEC, and ACC scores, I obtained compelling results compared with existing methods. Compared to the state-of-the-art algorithms, the WKFCM-DBF method\u2019s main advantages are better accuracy in retinal blood-vessel extraction of either poor or high quality and less running time. Moreover, based on the images and the table in"}
+{"text": "Chimeric antigen receptor T-cell therapy-related neurotoxicity is a novel cytokine-mediated neurological syndrome that may present with a broad spectrum of manifestations. Descriptions of novel distinctive features are pivotal to untangling this condition's clinical and instrumental signature in order to inform diagnosis and pathophysiology.A 27-year-old female patient received anti-CD19 CAR T cells for a refractory primary mediastinal B-cell lymphoma. At 6 days after the infusion, she developed mild ideo-motor slowing, dysgraphia, and drowsiness. Despite specific treatment with dexamethasone, her neurological status progressively worsened to a comatose state within 24 h. EEG and CSF analyses were non-specific, showing background slowing and inflammatory findings. Brain MRI revealed multiple focal punctate areas of T2-weighted hyperintensity localized in the body and isthmus of the corpus callosum. Following the administration of high-dose intravenous methylprednisolone, her neurological status resolved within 48 h. Notably, the follow-up brain MRI did not reveal any abnormalities in the corpus callosum, except for a reduction of fractional anisotropy.Reversible punctate inflammatory foci of the body and isthmus of the corpus callosum may represent a novel radiological finding of CAR T-cell therapy-related neurotoxicity. Chimeric antigen receptor (CAR) T-cell therapies are changing the treatment paradigms for B-cell malignancies by achieving durable complete remission in patients refractory to multiple lines of treatment . In contproducts. Descriptions of novel distinctive features are pivotal to untangling this condition's clinical and instrumental signature in order to inform diagnosis and pathophysiology. Most patients present with unremarkable brain MRI, yet a few recurrent neuroradiological features have been recognized , 9, 10. An otherwise healthy 27-year-old female patient was referred to our center for anti-CD19 CAR T-cell therapy (axicabtagene ciloleucel: axi-cell) for primary mediastinal B-cell lymphoma (PMBCL), stage IIXB at diagnosis for mediastinal bulky, refractory to two lines of standard chemotherapy . Before We described the case of a young woman affected by refractory PMBCL who displayed undescribed transitory inflammatory abnormalities of the corpus callosum during neurotoxicity related to CAR T-cell therapy. Our patient developed neurological manifestations on the seventh day from axi-cel infusion, subsequently to CRS resolution, and progressed to severe neurotoxicity within 48 h. Accordingly, ICANS median onset time is 4\u20136 days after CAR T-cell infusion, depending on CRS timing, and a rapid progression within a few h/days has been reported as the typical evolution . She preNotably, at the follow-up brain MRI, no T2-weighted hyperintensity was observable, yet DTI sequences detected microstructural abnormalities related to altered integrity of the white matte fibers of the corpus callosum . CollectReversible punctate inflammation of the body and isthmus of the corpus callosum may represent a novel radiological finding of CAR T-cell therapy-related neurotoxicity. Descriptions of novel distinctive investigative features are pivotal to defining a neuroradiological signature that would inform diagnosis and pathophysiology.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.UP and FP: drafting of the manuscript for content. FP, CP, DM, SM, LP, AS, and SB: revision of the manuscript for content, major role in the acquisition of data, and analysis or interpretation of data. UP, CP, and SB: study concept or design. All authors contributed to the article and approved the submitted version."}
+{"text": "This study evaluated 6\u2010month durability of antibody titers in LT recipients following two\u2010dose homologous mRNA vaccination, focusing on the impact of antimetabolite use.Anti\u2010spike antibody waning after two doses of severe acute respiratory syndrome coronavirus 2 (SARS\u2010CoV\u20102) messenger RNA (mRNA) vaccination among liver transplant (LT) recipients is anticipated, but the peak and decay kinetics might vary by patient phenotype.2, 3 Seroconversion was assessed using the Roche Elecsys anti\u2010receptor\u2010binding domain or EUROIMMUN anti\u2010S1 assays.Adult LT\u2010only recipients without reported SARS\u2010CoV\u20102 infection who had antibody titers measured at 1 month and 3\u20136\u00a0months following homologous mRNA vaccine series (D2) were included from a national observational study (IRB00248540).1 SeroPeak and longitudinal anti\u2010S1 trajectories were compared using multilevel mixed\u2010effects linear regression with a patient\u2010level random intercept and an interaction between mycophenolate use and time following D2.Between July 1, 2021, and October 26, 2021, a total of 161 participants received BNT162b2 (47%) or mRNA\u20101273 (53%) vaccination. Median (interquartile range [IQR]) age was 60 (46\u201367) years, and most were female (56%). Median (IQR) years since transplant was 6 (3\u201316). Peri\u2010vaccination immunosuppression regimens included calcineurin inhibitor (87%), antimetabolites (38%), corticosteroids (22%), and mammalian target of rapamycin inhibitor (16%); 20% or 7% received dual  or triple immunosuppression , respectively.Overall, 136 of 161 (84%) tested seropositive at a median (IQR) of 30 (28\u201332) days after D2. Of those with available paired titers, 133 of 149 (89%) were seropositive at 3 months, and 49 of 58 (84%) were seropositive at 6 months following D2. Of the 7 seronegative persons with paired titers, 4 (57%) seroconverted to low\u2010level positive titer by 6 months.4p\u00a0<\u20090.001) and 3 months  following D2. Additionally, participants taking mycophenolate had lower peak antibody levels , albeit similar rate of decay by 6\u00a0months following D2  ; the Pearl M. Stetler Research Fund from the Johns Hopkins School of Medicine; and the National Institute of Allergy and Infectious Disease . The analyses described here are the responsibility of the authors alone and do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government."}
+{"text": "Biomolecules called \u201cToxic and Essential Metals in Human Health and Disease 2021\u201d represents a follow-up of the previous Special Issue with the name of \u201cToxic and Essential Metals in Human Health and Disease\u201d. We intended to further explore the physicochemical and biological aspects of the elements, both in terms of the expanding appreciation of the essentiality of individual elements and the toxicity of excessive amounts of each element. The nutritional importance and metabolic complexities of the trace and mineral elements are reflected by the diversity of conditions recognized as being affected by derangements or deficiencies of these substances, such as neurodegenerative diseases, dermatological conditions, carcinogenic processes, osteoporosis, cardiovascular diseases, and endocrine dysfunctions. The ability of some trace elements to interfere with the biochemical and physiological utilization of molecular oxygen continues to sustain considerable research interest.The Special Issue of During the past decades, we have witnessed remarkable advances in the field of trace element research. Advances in analytical techniques have made it easier to measure concentrations of essential and of non-essential elements with great accuracy in biological fluids and samples. Among the current challenges today are the insufficient nutritional availability of iron, zinc and selenium in significant segments of the human population. At the same time, increasing urbanization poses an increased burden to population groups regarding some toxic elements, such as cadmium and mercury. Furthermore, among the intriguing but still unanswered questions are the molecular mechanisms underlying the apparent cardioprotective and anticarcinogenic properties of selenium.In the present Special Issue, sixteen contributions were published, including six reviews and ten original research articles spanning an extensive series of essential and non-essential metals.In their research article that was selected as Editor\u2019s Choice, Adamson et al.  present 3Hg+, Mg2+, Ca2+, and Sn2+. For the compound CH3Hg+, their speciation model identified the formation of the ternary complex CH3Hg-L-Cl. Upon further evaluation of the sequestering ability of dopamine towards the investigated cations, they disclosed the following order of affinity: Sn2+ > CH3Hg+ > Ca2+ > Mg2+. Their observations imply relevance also for in vivo effects of dopamine.Gigliuto et al.  studied,Alexandra Kuzan and coworkers  present In a review, Alehagen et al.  discuss Trypanosoma cruzi infection. Experimental and clinical data indicate that selenium may be used as a complementary therapy to prevent heart failure. The pathophysiology of Chagas cardiomyopathy appears to involve a complex immunological interplay involving the multifunctional cytokine TGF-\u03b2, the latter mediator operating as regulator of the immune response and the precipitated fibrosis. Selenoproteins induced by selenium supplementation could act as antioxidants to regulate the increased reactive oxygen stress present in the inflammation accompanying Chagas cardiomyopathy.Tania C. Araujo-Jorge et al.  present Tinkov et al.  present Bj\u00f8rklund et al.  review tSigrun Henjum and coworkers  have stuYasuda et al.  present Nordberg and Nordberg  review tAaseth et al. , in a reIn recent years, the mechanisms of Mn-induced neurotoxicity have been extensively studied by the research group of Michael Aschner . The neuIn a research article, Viviana Poliseno et al.  present 3 levels. In males, cold water swimming decreased PTH and resulted in a decrease in phosphorus content in bones, and an increase in 1,25-dihydroxyvitamin D3, and it may have positive consequences for the prevention of elderly sarcopenia.Bosiacki et al.  present 6 to 107 M\u22121s\u22121. Together, these observations suggest that the labile iron pool represents a constitutive peroxynitrite reductase system, as observed in their model of murine macrophage cells (RAW 264.7).Condeles and Toledo Junior  present Candida albicans. The transporter is localized in the plasma membrane and contains eight transmembrane domains corresponding to the amino acid sequence 126\u2013215. This amino acid sequence is considered to contain three metal binding motifs. Model peptides were studied to elucidate coordination properties of their Zn2+ and Cu2+ complexes, with the aim of identifying the most effective metal binding site among the three fragments. In the native Zrt2 protein, the Ac-GPHTHSHFGD-NH2 region of the Zrt2 loop had the highest metal binding affinity, demonstrating that three alternating histidines separated by only one residue bind Zn2+ and Cu2+ more strongly than the region in which three histidines are separated by two and three residues. All studied Zrt2 loop fragments have lower affinity towards Zn2+ than the zinc(II) binding site on the Zrt1 transporter. In addition, all three Zrt2 regions bind Zn2+ and Cu2+ with comparable affinity below pH 5 and, therefore, may equally contribute to the metal acquisition under the most acidic conditions in which the Zrt2 transporter is expressed.Belotti et al.  report s"}
+{"text": "Parasitic diseases pose a significant threat to global public health, particularly in developing countries. Host genetic factors play a crucial role in determining susceptibility and resistance to infection. Recent advances in molecular and biological technologies have enabled significant breakthroughs in understanding the impact of host genes on parasite adaptation. In this comprehensive review, we analyze the host genetic factors that influence parasite adaptation, including hormones, nitric oxide, immune cells, cytokine gene polymorphisms, parasite-specific receptors, and metabolites. We also establish an interactive network to better illustrate the complex relationship between host genetic factors and parasite-host adaptation. Additionally, we discuss future directions and collaborative research priorities in the parasite-host adaptation field, including investigating the impact of host genes on the microbiome, developing more sophisticated models, identifying and characterizing parasite-specific receptors, utilizing patient-derived sera as diagnostic and therapeutic tools, and developing novel treatments and management strategies targeting specific host genetic factors. This review highlights the need for a comprehensive and systematic approach to investigating the underlying mechanisms of parasite-host adaptation, which requires interdisciplinary collaborations among biologists, geneticists, immunologists, and clinicians. By deepening our understanding of the complex interactions between host genetics and parasite adaptation, we can develop more effective and targeted interventions to prevent and treat parasitic diseases. Overall, this review provides a valuable resource for researchers and clinicians working in the parasitology field and offers insights into the future directions of this critical research area. The prevalence of parasitic infection in tropical and subtropical regions is more severe owing to humidity and relatively lagging economic and sanitary conditions ; thus, p22.1https://www.ncbi.nlm.nih.gov/pubmed/) or Web of Science . After removing duplicated articles, we screened the titles and abstracts of the articles according to the following inclusion criteria: (1) studies published in English and (2) articles describing the differences among parasite migration, development, survival and pathogenicity caused by host genetic backgrounds. The exclusion criteria were as follows: (1) studies that were duplicates of graduate theses; (2) studies of influencing factors with no significant differences in hosts of different genetic backgrounds; and (3) studies not describing or related to parasite-host adaptation.In this study, we reviewed scientific studies with no time restriction to identify studies focusing on the factors of host genetic backgrounds that affect parasite-host adaptation. A total of 19 parasites whose adaptation has been documented were included in this study, including protozoans, flagellates, sporozoans, trematodes, cestodes and nematodes. Search terms were the combinations of \u201cLatin name for parasite\u201d, \u201chost\u201d, \u201chost susceptibility\u201d, \u201chost resistance\u201d, and \u201chost adaptation\u201d using the online databases PubMed (2.22.2.1Entamoeba histolytica (E. histolytica) is the only intestinal amebiasis known to cause amebiasis in humans, characterized by intestinal damage and amoebic liver abscess. Humans are permissive hosts for E. histolytica. Monkeys, cats, dogs, and mice can also serve as incidental hosts but retain natural resistance to amoebae. Some rodent strains, such as C3H/Hej, CBA/J, Mongolian gerbils, and hamsters, develop amoebic liver abscesses similar to humans, while others, such as BALB/c, C57BL/6 and rats, are somewhat resistant to the development of amoebic liver abscesses, which is attributed to the increasing levels of nitric oxide (NO) and some higher levels of cytokines, such as IL-10  mice show no difference from female mice in the size of amoebic liver abscesses as a consequence of the decrease in Th17 cells and cytokine IL-6 located downstream  is the agent of cutaneous leishmaniasis (CL), the most prevalent clinical form of leishmaniasis in Brazil. However, only a few infected people exhibit symptoms. The underlying mechanism varies but is genetically correlated. First, a single nucleotide polymorphism (SNP) is involved. As reported, people carrying the rs2275913 A SNP of the IL17A gene are more vulnerable to L. braziliensis infection than those carrying the rs2275913 G SNP (Leishmania infection than wild-type (WT) mice, likely by suppressing the Th1 response. Conversely, the enforced Th1 response led the IL-18 knockout BALB/c background mice to exhibit much resistance to Leishmania infection(Leishmaniosis is a zoonosis caused by ustelids . Leishma13 G SNP . For car13 G SNP . The effinfection and onlyinfection exhibit infection. IL-1\u03b1 iinfection. The lesinfection. IL-22 iinfection. The geninfection. The IL32.3.2Trypanosoma in humans includes Trypanosoma brucei gambiense (T. b. gambiense), Trypanosoma brucei rhodesiense (T. b. rhodesiense) and Trypanosoma cruzi (T. cruzi). T. b. gambiense and T. b. rhodesiense are prevalent in Africa and are transmitted by the Glossina palpalis and Glossina tsetse flies, respectively. The reservoirs of T. b. gambiense include humans, cattle, pigs, sheep and dogs, whereas those of T. b. rhodesiense comprise humans, sheep, cattle, lions and dogs  haplotypes, which affect their antigen presentation and recognition. As is known, C57BL/6 mice have the H-2b haplotype, which is more efficient in presenting peptides derived from intracellular pathogens, whereas BALB/c mice have the H-2d haplotype, which is more efficient in presenting peptides derived from extracellular pathogens. And this genetic background difference results in their different susceptibility to T. cruzi. Moreover, In B10 or DBA/1 mice, the H-2q haplotype is related to resistance, whereas the C3H genetic background is related to susceptibility  is a parasite infecting the human vagina and urinary tract, where it causes inflammation. Hitherto, it has been believed that T. vaginalis causes disease only in humans , Plasmodium. vivax (Pv), Plasmodium. knowlesi (Pk), Plasmodium. malariae (Pm), and two subspecies of Plasmodium ovale (Po)  . Among tcognized . Althougifferent . Furthert humans . On the t humans . The afft humans . In addit humans .Plasmodium can be divided into hosts with intermediate host adaptation and hosts with definitive host adaptation. Humans are a special intermediate host, which is mainly represented in the fact that humans in some populations are naturally resistant to malaria. The ligand-receptor pair composed of Duffy-binding protein (DBP-RII) on the surface of Pv and Duffy antigen receptor chemokine (DARC) on the surface of erythrocytes is an essential molecular basis for infection. The surfaces of the erythrocytes of most black people in West Africa lack DARC because of natural selection, so these individuals are naturally resistant to Pv  is an obligate intracellular and opportunistic parasite that parasitizes all karyocytes and mainly spreads by peroral infection and vertical transmission. The life history of T. gondii is divided into the sexual stage  and the asexual stage . Only cats and felines are definitive hosts for T. gondii . The higher level of NO in rats is more resistant to T. gondii when than that in mice  and Cryptosporidium hominis (C. hominis). In addition, several other Cryptosporidium species also infect patients with immunodeficiency diseases such as acquired immunodeficiency syndrome (AIDS)  . As an oII types . A high n adults . Moreoveaptation . In the aptation . The genaptation . Howeverporidium .2.52.5.1C. sinensis comprise the first intermediate host (freshwater snails), second intermediate hosts (freshwater fish and shrimp) and definitive hosts. More than 30 mammals, including humans, serve as definitive hosts  comprise the intermediate host (Lymnaeidae) and definitive hosts . Differences in genetic background among intermediate hosts have notable influences on susceptibility to F. hepatica  and uncommon hosts . The adaptation of F. hepatica varies in different definitive hosts. In uncommon hosts, the egg size is smaller than that in common hosts, and the ability of eggs to develop into miracidia is weaker, thus leading to a reduction in their prevalence , leading to the stronger susceptibility of BS-90 strains to S. mansoni. In addition, the inhibition of HSP 90 reverses the susceptible phenotype of NMRI strains  and NO, determined by the genetic background  is analogous to that of Hymenolepis diminuta (H. diminuta). Mice and humans serve as their definitive hosts, in which adult worms parasitize the intestine. Similar to most parasites, NO presents obvious inhibition capacity to hymenolepis  comprise intermediate hosts and definitive hosts. Artiodactyls , horses, kangaroos, rodents, primates and humans are intermediate hosts that can be infected by the hydatid cyst of E. granulosus and develop echinococcosis , to which hydatid cyst laminated layer glycans directly bind  mouse strains are more susceptible to T. trichiura, while H-2b (B10) or H-2q (B10.G) background strains are more resistant. Host sex also affects resistance to T. trichiura via a hormone-regulated immune response , Ancylostoma canis (A. canis) and Ancylostoma brasiliensis (A. brasiliensis), also infect humans under certain circumstances. Hookworms manifest obvious infection diversity in hosts  is a species of facultative parasites, and the life cycle includes free-living and parasitic generations. The hosts of parasitic generations comprise humans, dogs, cats, gerbils and mice  is one of the most frequent parasites of carnivorous mammals  can develop into sexual maturity in BALB/c mice, and the infectious microfilariae can be released into the surrounding environment; however, in C57BL/6 mice, the worms are eliminated before microfilariae are released  remains one of the leading causes of eosinophilic meningoencephalitis worldwide. Infections often occur through the ingestion of food or contaminated water containing third-stage larvae  and nonpermissive hosts   . In perms, etc.) . In nonps, etc.) .A. cantonensis fails to move from the cerebrum to pulmonary tissue in nonpermissive hosts such as humans, parrots and Mongolian gerbils, sporadic cases with worms present in the lungs are still observed. In humans, the worms were observed in both the brain and lungs of a male infant infected with A. cantonensis (A. cantonensis are also found in the lungs of parrots and Mongolian gerbils (A. cantonensis. In addition, the perturbation of these factors might affect the migration and development of worms in hosts. When fifth-stage larvae from the brains of the permissive host rats were transferred into the brains of the nonpermissive hosts rabbits or mice, no worms could migrate to the lungs (A. cantonensis larvae acquire the ability to migrate from the brain to the lungs but fail to develop sexually, indicating that larval migration in hosts occurs in an immunity-dependent manner, yet development relies on other factors (Although it is generally acknowledged that tonensis . In addi gerbils . Such exhe lungs , suggest factors .3Parasites remain one of the most threatening pathogens to humans worldwide. A promising strategy to control parasitosis is to intervene in deleterious parasite-host adaptation, which comprises parasite migration, development, survival and pathogenicity in hosts as well as host susceptibility and resistance to parasites. As a consequence of the distinct genetic backgrounds of hosts, after infection, parasite-host adaptations vary and result in different outcomes. In this article, parasite-host adaptation closely correlated with host genetic background-related factors and the underlying mechanisms were reviewed. In summary, various genetically related factors, including hormones, NO, immune cells, cytokines, genetic polymorphisms and metabolites, affect parasite-host adaptation Figure\u00a01To further explore the impact of parasite-specific receptors and host metabolites on parasite adaptation, in future studies, a combination of molecular and biochemical approaches can be used. CRISPR/Cas9 technology can be used to knock out or overexpress specific host receptors and investigate their impact on parasitic infection. Metabolomics can also be employed to identify host-derived metabolites that affect parasite growth and development. Furthermore, the utilization of patient-derived serum for the diagnosis and treatment of parasitic diseases can be a promising strategy. Investigating the specific antibodies and immune molecules present in patient serum that contribute to parasite resistance can aid in the development of targeted therapies for parasitic diseases.To further understand the complex mechanisms underlying parasite adaptation, in future studies, a multidisciplinary approach, integrating genetics, immunology, biochemistry, and ecology, can be employed. Combining genetic studies with ecological studies can provide insights into how host-parasite coevolution shapes the genetic diversity of both hosts and parasites. In addition, the integration of immunological and biochemical studies can elucidate the molecular mechanisms underlying the impact of host immune cells and molecules on parasite adaptation. Furthermore, the study of the impact of parasite adaptation on host physiological and behavioral traits can provide insights into the broader ecological consequences of parasitic infections. Investigations on the impact of parasite adaptation on host microbiota can also provide insights into the complex interactions among parasites, hosts and the microbiome.In summary, through an understanding of the underlying mechanisms of parasite-host adaptation from the perspective of host genetic backgrounds, it is expected that promising candidate targets to block the survival and development as well as the pathogenicity of parasites will be obtained. Interventions targeting key factors involved in host adaptation, enhancing the antiparasite immune response, exploiting patient-derived serum, blocking parasite-specific receptors, or interfering with the development and survival of parasites through inhibitors and diet restrictions, could ultimately achieve further advances in parasitosis control and therapy and deepen the understanding of the adaptive differences among parasites in different hosts.HZ and JL conceived this study and revised the manuscript. CY, LZ, LT, and YD undertook the literature review and drafted the manuscript. All authors contributed to the article and approved the submitted version."}
+{"text": "Laryngeal abscesses are rare in the modern antibiotic era. This report presents a novel case of an epiglottic abscess in a patient with diabetes who developed respiratory distress and was managed by awake intubation in the emergency room followed by transoral incision and drainage of the abscess and tracheostomy. Full recovery after 1\u00a0week of intravenous antibiotic treatment was observed. Surgical intervention is necessary for treatment and culture-directed antimicrobial therapy. Poorly controlled diabetes is a newly described risk factor for spontaneous epiglottic abscess development. Acute laryngeal infections with abscess formation are traditionally divided into primary and secondary infections. The former usually starts after precipitating events such as trauma or localized bacterial infection including Hemophilus influenza type B (HiB)-related epiglottitis, whereas the latter occurs when an infection from tonsillitis or nasopharyngitis spreads to the larynx . An epigAirway obstruction is the most feared complication of epiglottitis, whereas descending necrotizing mediastinitis and epiglottic abscesses are unusual complications that may necessitate surgical intervention . LaboratA 72-year-old man with diabetes presented to the emergency department with dyspnea, stridor and odynophagia that suddenly started 3\u00a0h earlier. Clinical examination revealed that the patient was febrile with hypoxia, hoarseness and drooling. Flexible fiber optic rhinopharyngo-laryngoscopy showed pooling of secretions in the pyriform sinuses, a swollen and edematous epiglottis, poor visualization of the glottis and reduced vocal cord movement. A lateral neck radiograph revealed a swollen epiglottis and, due to his respiratory distress, the patient was intubated by the emergency physician in the emergency room; CT of the neck with intravenous contrast revealed a multiloculated abscess involving the epiglottis, and paraglottic as well as parapharyngeal spaces . Laboratet\u00a0al. and Ridgeway et\u00a0al. [Streptococcus and Staphylococcus species are the most commonly identified causes of adult epiglottic abscesses [Neisseria meningitidis and Aeromonas hydrophila [Epiglottic abscess is a rare disease in the modern era, owing to the availability of a wide range of antibiotics and modern investigation techniques . It repry et\u00a0al.  as a risbscesses , patientdrophila . In the Fever, sore throat, odynophagia, dysphagia, neck discomfort, stridor, drooling and shortness of breath are common signs and symptoms of acute infections, as observed in our case. Patients are at risk of rapid airway compromise. Therefore, monitoring patients for signs of oxygen desaturation, worsening stridor, adoption of a tripod posture and drooling is crucial .Different investigative modalities can be used if a patient does not experience severe respiratory distress. Flexible fiberoptic laryngoscopy is a quick, safe and accurate method to quickly assess the supraglottic and glottic areas as well as airway patency. A lateral neck soft tissue radiograph showed an enlarged epiglottis (thumb sign), which can also be observed in acute epiglottitis. CT scan of the neck with intravenous contrast is helpful in evaluating the airway and differentiating acute epiglottitis from epiglottic abscess; accordingly, it represents the best modality for accurate diagnosis and follow-up of the condition . SimilarThis case highlights the importance of a multidisciplinary approach in patient care as well as the use of various diagnostic and therapeutic techniques to manage epiglottic abscesses. Airway control was performed in an operating room. Awake intubation using a video laryngoscope, bronchoscopy-guided intubation or tracheostomy under local anesthesia are all possible airway management strategies . After sAntibiotics with wide coverage should be selected; a combination of third-generation cephalosporins and metronidazole provides coverage against Gram-positive, Gram-negative and anaerobic bacteria . SteroidIn conclusion, acute laryngeal abscesses are considered uncommon in the current era due to vaccination efforts, novel antibiotics and early diagnosis of epiglottitis. This case serves as a crucial reminder for physicians to check for epiglottitis in patients presenting with these symptoms to prevent the progression of epiglottitis into an epiglottic abscess. Early diagnosis and treatment of these manifestations can reduce the risk of airway loss, which can be fatal. Regular monitoring of blood glucose levels and good diabetes control can help reduce the risk of epiglottic abscess development and accelerate recovery."}
+{"text": "Glioblastoma (GBM) is among the most fatal and recurring malignant solid tumors. It arises from the GBM stem cell population. Conventional neurosurgical resection, temozolomide (TMZ)-dependent chemotherapy and radiotherapy have rendered the prognosis of patients unsatisfactory. Radiotherapy and chemotherapy can frequently induce non-specific damage to healthy brain and other tissues, which can be extremely hazardous. There is therefore a pressing need for a more effective treatment strategy for GBM to complement or replace existing treatment options. Cell-based and cell-free immunotherapies are currently being investigated to develop new treatment modalities against cancer. These treatments have the potential to be both selective and successful in minimizing off-target collateral harm in the normal brain. In this review, several aspects of cell-based and cell-free immunotherapies related to GBM will be discussed. According to the 2016 World Health Organization (WHO) histopathological and clinical criteria, gliomas are classified as grades I-IV . In contGlioblastoma (GBM) has been described as a grade 4 tumor by the WHO and is among the most fatal and recurring malignant solid tumors to date  accountiAs early as the mid-nineteenth century, it was proposed that cancer treatment could be achieved by modulating the body\u2019s immune system to combat cancer . Its dis22.1in vivo persistence (Chimeric antigen receptor (CAR) T cell therapy holds one of the most promise as an anti-cancer therapeutic technology. CAR are synthetic molecules formed by four regions, the antigen recognition structural domain , the extracellular hinge region, the transmembrane structural domain (consisting of a hydrophobic \u03b1-helix across the cell membrane) and the intracellular T cell signaling structural domain   intendedsistence . Third gsistence . Fourth sistence . The fifsistence Figure\u00a02CAR-T cells were first used to treat hematological malignancies and have shown remarkable efficacy , 22. For2.1.1IL-13R\u03b12 as a target for CAR-T in the treatment of GBM. It\u2019s a membrane receptor with a high affinity for the anti-inflammatory cytokine interleukin 13 and it has been discovered to be overexpressed in a variety of solid tumors, most notably GBM, and has been related to poor prognosis . IL-13R\u03b1in situ mouse models, this approach has not yet been validated in patients. In 2015, they published the first promising human clinical study of intracranially administered IL13R\u03b12-specific CAR-T cells for GBM, which set the stage for the future application of improved peripatetic CAR-T cells therapy , as the first tyrosine kinase receptor to be cloned, is still at the leading edge of targeted cancer therapy. Being the most common variant of EGFR, EGFRvIII is usually expressed in GBM  and is ain situ models of human EGFRvIII+GBM, EGFRvIII-targeted CAR T cells were also able to suppress tumor development. They also planned a phase I clinical research utilizing humanized scFv-transduced CAR T cells targeting EGFRvIII in patients with residual or recurrent GBM based on these findings   targeting EGFRvIII mutant oncoproteins is a therapeutic approach , and sec2209376) . A first2209376)  in whichlization , hence tlization  used a t2.1.3in vitro with the aim of identifying and killing EphA2-positive glioma cells and glioma-initiating cells, as well as inducing tumor induction in an in situ xenografted GBM with Severe Combined Immunodeficiency (SCID) mouse model of regression. However, such manipulations are still carried out through highly artificial means and may not better predict future clinical effectiveness. H.T. Lin et\u00a0al.  for broader application. Trivalent CAR-T cells have the potential to overcome antigen heterogeneity in GBM and improve treatment results. Furthermore, Joseph H. Choe et\u00a0al.  as a delivery vehicle based on NEs to effectively deliver PTX to tumour cells and induce cytotoxicity and inhibit post-operative recurrence of GBM , the most prevalent leukocyte population in the blood, may be rapidly recruited to sites of inflammation, and are thought to be a powerful platform for tumour-targeted drug delivery, similar to mesenchymal stromal cells (MSCs) . More ime of GBM . Furthere of GBM  internale of GBM \u201363. The henotype . To addrhenotype  mounted 2.3MSCs are pluripotent stem cells, which are normally derived from bone marrow, umbilical cords/placenta, and adipose tissue. MSCs have tissue healing capability and low immunogenicity, and they are not restricted by the BBB/BTB, so they can be intrinsically subsumed into the brain tumour site , 65, ove2.3.1in vivo situation where tumour cells are integrated with supportive cells more unknown. Improving the efficiency of MSCs in the treatment of GBM requires the use of appropriate tools and technical abilities. Extensive research has revealed the promise of suicide genes in the treatment of glioma tumors. Enhancing their effectiveness relies on the ability to apply the right tools and techniques. Saeed Oraee-Yazdani et\u00a0al.  in GBM treatment affected by the rapid clearance and non-specific bio-distribution of gadolinium-based drugs, Yen-Ho Lai et\u00a0al.  and chemokines  to better recruit MSCs to GBM sites , 98. In on model . OV seleon model . The useon model . Delta-2on model . MSCs loon model . MSCs enon model .2.4in vitro destruction by healthy allogeneic NK cells. Their findings demonstrated that GBM tumour-infiltrating NK cells acquired an altered phenotype associated with impaired lytic function when compared to matching peripheral blood NK cells from GBM patients or healthy donors. This immune evasion approach was attributed to \u03b1v integrin-mediated TGF-\u03b2 activation, which directed interactions between GSCs and NK cells. In contrast, blocking the \u03b1v integrin/TGF- \u03b2 axis can increase NK cell antitumor function. Gregory J Baker et\u00a0al.  cells play an essential role in the body\u2019s anti-infection, anti-tumour, and immunomodulatory processes as recognition and effector cells in the innate immune system. NK cells do not inhibit the killing of their own normal cells, but selectively recognize and kill cells that are low in expression or lack their own major histocompatibility complex (MHC)-I molecules . MHC-I mr et\u00a0al.  found thin vitro and in vivo. In contrast to CAR-T cells, which require autologous cells for each patient, NK cells are safe under allogeneic circumstances, which broadens the pool of cell donors capable of producing therapeutically meaningful amounts of CAR-NK cells for therapy  are now recognized as the most potent and only specialist antigen-presenting cells in the body capable of activating na\u00efve T cells. It is called after the numerous dendritic protrusions that emerge from the cell surface during maturation. Immature and mature dendritic cells perform distinct tasks, with immature dendritic cells being good at antigen differentiation and phagocytosis and mature dendritic cells being good at antigen presentation. DC have MHC class I and II molecules on their surface, which when combined with antigenic peptides trigger CD4+ helper T cells (Th) and CD8+ cytotoxic T lymphocytes (CTL) to elicit specific anti-tumour cellular and humoral immunity, inhibiting tumour cell growth . DC alsoClinicalTrials.gov, NCT01280552). HLA-A2 primary tumour antigen expression was more frequent than in HLA-A1 patients. HLA-A2 patients had a higher immune response (by Elispot) and patients in the pre-specified subgroups of methylated and unmethylated MGMT achieved meaningful therapeutic benefit with ICT-107. This was the first vaccination study to demonstrate a clinical benefit in GBM, and it paved the way for a phase III trial in patients with HLA-A2+ newly diagnosed GBM. Linda M Liau et\u00a0al.   Table 2.0045968) . The use0045968) , 134. Th2.6via the impaired BBB/BTB, and both cells are converted into critical drivers of tumour development by acting as TAMs together to infiltrate at GBM locations (Microglia and tumour-associated macrophages (TAMs) are the main components of GBM myeloid cells, which are maintained by self-renewal under physiological conditions and are associated with functions such as CNS inflammation and development . Under pocations .TAMs are the most common immune cells in the TME, and their phenotype is diverse and flexible . The bul33.1Despite the many advantages of MSCs in the fight against GBM, there are some limitations to the use of MSCs, such as low biological activity and limited accessibility , 80, 87.MSCs can be an abundant source of Exo, and all Exo express the same group of proteins, such as tetraspanins , adhesion proteins , Alix and TSG101 \u2013150. ExoMSCs exosomes (MSCs-Exo), as paracrine mediators of the therapeutic effect of MSCs, have comparable biological activity to MSCs. However, compared with MSCs, MSCs-Exo are smaller, penetrate biological membranes more easily, are less immunogenic, more biocompatible, and better preserved , 156. Pr3.2via several mechanisms. At first, viruses proliferate in tumour cells and directly lyse tumour cells  have become effective new therapeutic tools in this field \u2013182. OV ur cells . Then, lur cells \u2013191. Dueur cells Table\u00a03.3.2.1Oncolytic herpes simplex virus (oHSV-1) is a neurophilic double-stranded DNA virus. Typically, wild HSV-1 is neurotoxic, so the virus must be genetically modified or greatly attenuated to ensure safety. After genetic modification, OV can still maintain its ability to reproduce while replicating specifically in tumour cells, and therefore OV is widely exploited. Based on previous findings, oHSV was the first state-of-the-art genetically engineered OV to be licensed by the United States FDA for cancer treatment  and was in vivo.However, as GBM is a primary brain tumour of the human central nervous system, more attention deserves to be paid to the safety of oHSV-1 in the fight against GBM. In past studies, oHSV-1 is encoded by the \u03b334.5 gene, an ICP34.5 protein, which is neurotoxic . To limiG207 is a second generation oHSV-1 that inactivates the ICP6 gene by deleting the double copy \u03b334.5 gene while inserting the lacZ gene at the UL39 locus. During a Phase I clinical trial of genetically engineered oHSV-1 G207 by GK Friedman et\u00a0al. , oHSV-1 G47\u0394 is a third generation oHSV-1 with a triple mutation in \u03b147 deleted from the G207 genome. Compared to G207, G47\u0394 is further attenuated in normal cells, but has enhanced efficacy in anti-tumour, as well as a greater safety profile. oHSV-1 G47\u0394 showed efficacy and safety in GBM was confirmed by the American Association for Cancer Research in 2016 . In subs3.2.2Adenovirus is a non-enveloped virus with an icosahedral capsid containing approximately 38 kb of genomic double-stranded DNA. There are more than 50 serotypes of adenovirus in humans, of which types 2 and 5 have been most frequently studied for the manufacture of lysing viruses , 206. Invia the integrins av\u03b23 and av\u03b25 into tumour cells, which further enhances tumour targeting  that degrades HA and spreads efficiently in the tumour. It has the same mechanism of action as Delta-24-RGD, ICOVIR17 deletion of 24 base pairs in the Rb-binding domain of E1A for tumour-selective replication and RGD modification in the fibril to amplify tropism, except for two additional modifications: insertion of an E2F binding site in the E1A promoter and insertion of the SPAM1 gene encoding PH20 HA after the fibril, which is controlled by the major late promoter control . Normallin vivo. Jawad Fares et\u00a0al.  , 217. Ths et\u00a0al.  conducte3.2.3Reovirus (RV) belongs to the wild-type OV family of eutheroviruses and is characterized as a staged double-stranded RNA virus. The three prototypical serotypes of RV, first identified in the 1960s, are well characterized as serotype 1 Lang (T1L), serotype 2 Jones (T2J) and serotype 3 Dearing (T3D) . RV is w3.3GBM generates an immunosuppressive environment through multiple mechanisms, including the programmed cell death protein 1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), lymphocyte activating gene 3 (LAG-3) pathway . AlthougThe immunosuppressive properties of the GBM microenvironment lead to immune evasion by tumour cells, making inhibition of immune checkpoints such as PD-1 alone ineffective , 227. PD3.3.16-methylguanine-DNA methyltransferase (MGMT) renders tumors resistant to TMZ, MGMT promoter status predicts both prognosis and therapeutic response to TMZ chemotherapy. Antonio Omuro et\u00a0al.  of HSV-1 against PD-1, which also expresses a single-chain fragment mutable antibody (scFvPD-1). Irene Appolloni et\u00a0al. , a triple combination that could assist in curing less immunogenic malignancies such as GBM (31). Also based on the fact that OV and PD-1 inhibitors have become standard immunotherapies against certain cancers, Carmela Passaro et\u00a0al.  conductei et\u00a0al.  also inv4There is a deepening perception of what cell-based and cell-free immunotherapy can bring to GBM, which offers new hope for GBM patients, but many details and questions remain to be explored and elucidated. According to recent statistics, adjuvant immunotherapy can prolong survival and significantly improve outcomes for patients with recurrent GBM compared to those treated with only surgery, radiotherapy or chemotherapy. To summarize the lessons learned, the extremely specific and heterogeneous nature of GBM, as well as the complicated immune resistance mechanisms and immunosuppressive TME have resulted in relatively limited response effectiveness and durability of response to treatment drugs. The BBB/BTB is another barrier to medication delivery. These are still tough breakthroughs in GBM therapy. We not only obtained some novel findings on the theoretical study of the local immune characteristics of glioma, but we also provided an experimental basis for the comprehensive diagnosis and treatment of regulating and intervening in the immune microenvironment of glioma. More significantly, \u201csupporting the righteousness\u201d and improving the immune microenvironment, in conjunction with \u201celimination of evil\u201d by anti-tumour cells, will ideally decrease disease mortality rates, extend patient life, and genuinely help patients. In fact, the treatment of GBM is actually a complex \u201cproject\u201d and satisfactory results can hardly be achieved with only one treatment. Immunotherapy research for GBM should be coupled with other treatment modalities in future anti-GBM research, resulting in truly tailored and complete care strategies for patients. It is recommended to develop combined treatment techniques based on immunotherapy, molecular targeted therapy, and radiation to maximize therapeutic efficiency and reduce acquired immunotherapy resistance. The industry therefore needs to innovate, integrate and translate to drive immune combinations forward in a sustained manner. Secondly, assessing therapeutic response to immunotherapy is difficult, and in the future, a standardized imaging and molecular biology evaluation system will be required to reflect and forecast patient outcomes. Cell-based and cell-free immunotherapy are predicted to become an essential component of future glioma treatment when these challenges are solved.MW conceived this article. MW and XW drafted the manuscript. MW and XW drew the illustrations and tables. MW, XW, XJ, JZho and YZ revised the article. MW, YL and JZha checked and edited the article. YL and YY supervised the article. All authors have read and agreed to the final version of the manuscript."}
+{"text": "GBM-on-a-chip platforms can integrate biological or chemical functional units of a tumor into a chip, mimicking in vivo functions of GBM cells. This technology has shown great potential for applications in personalized precision medicine and GBM immunotherapy. In recent years, there have been efforts to construct GBM-on-a-chip models based on microfluidics and bioprinting. A number of research teams have begun to use GBM-on-a-chip models for the investigation of GBM progression mechanisms, drug candidates, and therapeutic approaches. This review first briefly discusses the use of microfluidics and bioprinting technologies for GBM-on-a-chip construction. Second, we classify non-surgical treatments for GBM in pre-clinical research into three categories  and focus on the use of GBM-on-a-chip in research for each category. Last, we demonstrate that organ-on-a-chip technology in therapeutic field is still in its initial stage and provide future perspectives for research directions in the field.Glioblastoma (GBM) is the most malignant type of primary intracranial tumor with a median overall survival of only 14 months, a very poor prognosis and a recurrence rate of 90%. It is difficult to reflect the complex structure and function of the GBM microenvironment  It is tin vitro GBM models includingboth GBM tumor and normal brain tissues. However, traditional Petri-dish-based assays do not fully represent the complexity of tumors, limiting their potential use to determine predictive functional biomarkers. Organ-on-a-chip (OoC) is a revolutionary novel technology that has been developed rapidly in the past decade. Using OoC technology, human functional units constituting tissues and organs can be simulated ex vivo on microscopic cell and tissue culture vehicles, including the basic components and elements necessary for functional units, such as multicellular components, extracellular matrix (ECM) and physicochemical microenvironmental factors ; this technology is termed \u2018GBM-on-a-chip\u2019 , 17. GBMe manner . GBM-on-e manner .In this review, we present the microfluidics and bioprinting technologies that are currently used to construct GBM-on-a-chip models. We also review recent studies of the use of GBM-on-a-chip in a variety of treatments including chemotherapy, immunotherapy and other therapies . By describing the applications of these GBM-on-a-chip models in GBM investigation, we provide a broad perspective on the progress and future of the technology.22.1The TME is closely related to tumorigenesis, tumor development, and metastasis . In receOverall, GBM is closely linked to the GBM TME. GBM can release cell signaling molecules to influence the TME by promoting tumor angiogenesis and inducing immune tolerance, while immune cells within the TME influence GBM cell growth and development . Further2.2Most organ- or tumor-on-a-chip systems, such as GBM-on-a-chip models, are constructed using microfluidics technology, and the majority of microfluidic devices have been fabricated through photolithography and soft lithography , 29. Thein vitro fluid flow environment. The system was equipped with continuous nutrient circulation and waste removal, allowing for an average cultivation period of 72 h. The tissue viability as analyzed by flow cytometry was 61.1% in tissue maintained on the microfluidic platform after 72 h, compared with 68.9% for fresh tissue, demonstrating that patient-derived GBM tissue could be successfully maintained within the microfluidic chip to model biological processes and tissue structures of tumors for the mechanistic and therapeutic investigation in GBM. In another study, Dou et\u00a0al. used soft lithography to create a polyacrylamide hydrogel-based GBM model that could precisely generate orthogonal chemical stimulation and controllable stiffness gradients to investigate the biological behaviors of GBM cells , TMZ, and the Res + TMZ combination on GBM  . The chin on GBM . Comparen on GBM , 48. Jien on GBM . As effe3.1.3via a concentration gradient to regulate the release of chemicals. It also provided a large number of miniature culture chambers in which high-throughput GBM spheroids could be formed  hydrogel, to test a combination drug therapy consisting of Pitavastatin and Irinotecan . This ple formed . After 4e formed . New pyre formed . GBM celin vitro model using OoC technology that accurately represents the GBM TME, especially the BBB. Such patient-specific models could be used to screen the most appropriate drug combinations for individuals. However, owing to its lack of capacity to reflect neurotoxicity and other adverse effects on patients, the model would need to be integrated with multiple biological systems that can recapitulate the complex functionalities of different human tissues or organs so as to simulate the physiology of the patient with a high degree of fidelity. Thus, researchers could search for better chemotherapeutics to target GBM while reducing drug-induced injury.As GBM tumors inevitably recur after surgery and radiation treatment, chemotherapy plays an important part in killing the remaining GBM cells. However, the BBB prevents the entry of adequate chemotherapeutic drugs into the cerebral circulation brain, limiting the effects of systemic chemotherapy against GBM. To overcome this limitation, there is a need for a patient-specific 3.2GBM is highly heterogeneous, and extrinsic components of tumor cells that are inherent to the brain, as well as intrinsic mechanisms of tumor cells that assist immune evasion make the GBM TME extremely challenging to cope with . Reducin3.2.1TAMs can secrete a variety of enzymes, reactive oxygen species, growth factors, and cytokines that contribute directly and/or indirectly to tumor proliferation, invasion and angiogenesis in GBM . Thus, tvia STAT3 regulation, providing initial evidence for the use of miR-124 EVs to develop a novel therapeutic strategy. The miR-124 EV treatment also suppressed tumor progression and anti-tumor immune responses, leading to enhanced intratumoral infiltration of natural killer (NK) cells (via the integrin (\u03b1v\u03b23). Using this GBM-on-a-chip model, a novel dual \u03b1v\u03b23 and TGF-\u03b21 blockade was found to suppress tumor neovascularization of GBM by simultaneously targeting endothelial-macrophage interactions and macrophage-associated immunosuppression.Gu et\u00a0al. established three microfluidic assays, which they refer to as co-migration assays, based on a microfluidic device that can be used for the investigation of the bi-directional relationship between GBM cells and microglia . MicroglK) cells . The res3.2.2ICB therapy has achieved great success in the treatment of advanced tumors of various types, including melanoma, lymphoma, lung cancer, and kidney cancer, with significant improvements in median overall survival in recent years \u201369. ImmuCui et\u00a0al. developed a patient-specific GBM-on-a-chip platform for analyzing the heterogeneity of immunosuppressive TMEs and optimizing anti-programmed cell death protein 1  immunotherapy against various GBM subtypes . This pl3.2.3CAR-T therapy, a revolutionary cellular immunotherapy by which T cells are genetically modified, has been approved for specific haematological malignancies and shows potential to target a variety of solid tumors . EGFRvIIHuang et\u00a0al. created a microfluidic platform based on a transwell BBB and U87vIII co-culture system for assessment of BBB extravasation of U87MG cells expressing tumor-specific mutant protein EGFRvIII (U87vIII) targeted by CAR-T . ControlIn recent years, OoC technology has proved able to almost fully reproduce the GBM tumor immune microenvironment and has become a potent tool for investigation of GBM immunotherapy. However, as microfluidic chips are usually constructed using artificially engineered materials, they may not exactly replicate the real TME. Moreover, there are many geneogenous immunizing cells and adaptive immunizing cells, and the absence of one cellular component or incorrect cellular proportions may result in differences compared with the natural tumor immune microenvironment. Thus, standardization is urgently required to enable researchers to build homogenous models with standard methods that can reproduce the complexity of the GBM tumor immune microenvironment in the future.3.33.3.1Phototherapy comprises two main approaches: photodynamic therapy (PDT), and photothermal therapy (PTT). PDT can cause local chemical damage to target lesions under specific light irradiation, using a photosensitizer to produce large amounts of reactive oxygen radicals that can kill tumor cells. PTT causes local thermal damage when the photothermal agent is irradiated by light at a specific wavelength, causing it to heat up and consequently kill tumor cells . The usePDT requires three key elements, namely, a photosensitizer, oxygen, and light, to comprehensively improve its efficacy , 77. LouPTT uses the light-to-heat ability of photothermal agents to enhance the heating of cells and tissues in a localized region. Cell death occurs almost instantaneously owing to protein denaturation and destruction of plasma membranes at tissue temperatures greater than 60\u00b0C, which are usually reached with PTT . Lee et\u00a03.3.2in vivo. The MHT assay was performed after C6 cells had been 3D cultured in the chip for 48 h. MNPs consisting of magnetite coated with aminosilane were used to evaluate the efficacy of MHT in C6 cells. After 30 min of magnetic hyperthermia using the MNPs, nearly all GBM cells in the GBM-on-a-chip model were killed. (Magnetic hyperthermia therapy (MHT) is a novel type of anti-tumor physical therapy, that takes advantage of the thermal effects of magnetic nanoparticles (MNPs) with an alternating magnetic field (AMF) and the fact that tumor cells are less heat-tolerant than normal cells. The AMF is used to selectively kill tumor cells while MNPs are injected into the tumor site , 86. Mam3.3.3The technical principle of focused ultrasound (FUS) is to use ultrasound to penetrate human tissue without damage with a focus on the target lesion. This produces a thermal effect, force effect, and cavitation effect, resulting in direct or indirect regulation and treatment of the lesion area . UltrasoPhototherapy is a promising therapeutic option for cancer. To date, 5-Aminolevulinic acid-PDT has been approved by the Food and Drug Administration (FDA) for GBM treatment and has shown promising outcomes. However, its effectiveness is limited by the ability of the NIR laser to penetrate into deep brain regions. Therefore, future research should focus on increasing penetration depth in order to enhance the applicability of phototherapy. A number of challenges still need to be overcome before MHT can be applied to GBM clinical treatment, although there have been enormous advances in MHT research over the decades. For example, owing to a lack of specificity, MNPs could accumulate in healthy tissues as well as at the GBM tumor site, which might cause damage to surrounding structures. Moreover, MHT may not completely ablate the GBM tumor, leading to tumor recurrence. More research is also needed to provide sufficient clinical data to support its effectiveness and safety. A combination of phototherapy, MHT, and immunotherapy with an all-in-one microfluidic platform might be an option to achieve synergistic effects. Combined with FUS, drug-loaded microbubbles can temporally increase the permeability of the BBB and can be released at specific locations, enabling targeted delivery into the brain. However, there could be a sterile inflammatory response when the BBB is opened by FUS. In the future, emphasis should be placed on control of ultrasound parameters and the optimization of microbubble types and injection doses to achieve efficient drug delivery.4Bionic characteristics of the GBM TME, including cell-to-cell and cell-to-ECM interactions, capillaries, the BBB, and oxygen concentration gradients, can be reproduced by component construction and 3D cell arrangement with microfluidics and bioprinting in GBM-on-a-chip models. These models have considerable potential applications in studies of chemotherapy, immunotherapy, and other GBM therapies. GBM-on-a-chip models have been used to study the interactions between GBM cells and the brain microenvironment, demonstrating that GBM cells can alter the behavior of other cells in the brain, whereas the microenvironment can also influence the behavior of GBM cells. GBM-on-a-chip models have also been used to test the effects of different drugs and treatments on GBM cells in a more realistic microenvironment than those provided by traditional cell culture models. The efficacy of different drug delivery methods, such as nanoparticles and liposomes, can also be tested using GBM-on-a-chip models. However, the development of OoC technology in therapeutic fields is still in its initial stage. At present, GBM-on-a-chip models may not fully replicate the complex interactions between different cell types and the ECM that occur in the brain. Moreover, the models may not be able to capture the high heterogeneity of GBM that can vary greatly in terms of its genetic makeup and response to treatment, which could limit their usefulness in developing personalized treatment strategies for GBM patients. In the future, there is a need to build on the breakthrough of GBM-on-a-chip technology and develop more complex and bionic humanized GBM-on-a-chip models with more complex structure and function. There are still many new technologies in electrical and optical disciplines that can potentially be combined with GBM-on-a-chip, which would broaden the technical field of GBM therapy. For instance, optical-based bioprinting techniques enable rapid construction of GBM-on-a-chip models with continuous automated production. Combined with nanotechnology, GBM-on-a-chip platforms have the potential to regulate nanodrug delivery in response to electrical stimulation to facilitate targeted therapies, PPT, and PDT. Recently, the FDA has removed the requirement for animal testing prior to human clinical trials. This could represent an opportunity for OoC technology to usher in rapid development and replace animal models. One of the main advantages of OoC technology is that it can provide more accurate results than animal models. For example, a personalized GBM-on-a-chip platform can be used to develop patient-specific precision strategies and identify the best drug combination to optimize treatment outcomes in the broader GBM patient population. In the future, researchers could integrate GBM-on-a-chip with multi-organ chips to model the intersection of different biological systems. This could recapitulate organ-level physiology and pathophysiology of GBM patient, and leveraging computational modelling in combination with experimental data generated using this platform could lead to the development of effective new drugs with low side-effects and the discovery of novel therapeutic targets in GBM. As the technology continues to improve and become more widely adopted, it has the potential to transform the field of drug development and toxicology testing, while also reducing the need for animal testing.Conceptualization, JM and ZX; writing\u2014original draft preparation, ZX; writing\u2014review and editing, JM and HW; supervision, MC, and JF. All authors contributed to the article and approved the submitted version."}
+{"text": "To assess the anxiety and depression levels in patients with Posner-Schlossman syndrome (PSS) and to determine the potential risk factors.In this cross-sectional study, a total of 195 participants, including 93 PSS patients and 102 healthy controls were recruited. Sociodemographic and clinical information were collected for all participants. Hospital Anxiety and Depression scale (HADS) was administered to evaluate the anxiety and depression levels. Visual function (VF) and quality-of-life (QOL) questionnaires were administered to assess variables potentially associated with anxiety and depression.P\u2009=\u20090.009). While the frequency of depression between the two groups was not significantly different (P\u2009=\u20090.349). The mean anxiety and depression scores were 6.98\u2009\u00b1\u20094.20 and 6.44\u2009\u00b1\u20093.66 in PSS patients as compared to 6.67\u2009\u00b1\u20093.21 (P\u2009=\u20090.564) and 5.96\u2009\u00b1\u20092.93 (P\u2009=\u20090.311) in controls. Logistic regression analysis showed mental well-being was significantly associated with anxiety  and depression  in PSS patients.Increased anxiety level was observed in 22 (23.7%) PSS patients as compared to 10 (9.8%) of controls (More patients with PSS may experience anxiety as compared to healthy controls. Mental well-being is an independent risk factor for anxiety and depression. It is important for ophthalmologists to be aware of these factors and should pay more attention on mental health when PSS is managed in clinic.The online version contains supplementary material available at 10.1186/s12886-023-03047-4. Posner-Schlossman syndrome (PSS), also known as glaucomatocyclitic crisis, is characterized by unilateral recurrent episodes of acute elevated intraocular pressure (IOP) and mild non-granulomatous anterior uveitis , 2. ChroMany ocular diseases including glaucoma , Behect\u2019The psychological effects of ocular diseases are insidious and often underestimated . On the Thus, the purpose of the present study was to determine and compare the anxiety and depression levels in patients with PSS and healthy controls. The study, additionally, evaluates the sociodemographic and clinical factors that may contribute to anxiety and depression in patients with PSS.In this cross-sectional study, PSS patients were recruited based on the diagnosis of their medical files from Department of Ophthalmology of the Eye & ENT Hospital from January 2018 to December 2019. Healthy controls were age and gender matched healthy volunteers. PSS diagnosis was based on the following clinical features: (a) characteristic recurrent mild, unilateral, non-granulomatous anterior uveitis; (b) transient episode of elevated IOP with blurred vision; (c) discrete, round, white nonpigmented keratic precipitates (KPs); (d) deep anterior chamber with wide and open angle; (e) no posterior synechiae and inflammation [This study was approved by the ethics committee of the hospital and was conducted according to the Declaration of Helsinki. Written informed consent was obtained from all participants.General information including gender, age, alcohol drinking status and smoking status, along with clinical information including the number of eyedrops used and duration of disease were recorded.Two questionnaires were used in this study. 1) A Chinese version of the Hospital Anxiety and Depression Scale (HADS) , which h A Chinest test, whereas categorical variables were presented as counts and percentages and were compared using the Pearson \u03c72 test, or the Fisher exact test. Both univariable and multivariable logistic regression analyses were performed to determine the risk factors of anxiety and depression in PSS patients. A P-value\u2009<\u20090.05 indicated statistical significance.Data analyses were performed using SPSS . Continuous variables were reported as mean and standard deviation (SD) and were compared using the independent sample A total of 195 participants who met the inclusion and exclusion criteria were enrolled in this study, and the demographic and clinical characteristics were presented in Table\u00a0P\u2009>\u20090.05). Within each group, there were more participants who do not smoke or drink. In the PSS group, there were more male patients (57.0%) than female patients (43.0%).Out of the 195 study participants, there were 93 PSS patients and 102 healthy control participants. The age, gender, smoking and alcohol drinking status, VF total scales, QOL total scales, HADS-A scores and HADS-D scores between PSS patients and healthy controls showed no statistically significant differences , PSS patients reported significantly lower mental well-being (78.3\u2009\u00b1\u200919.4) as compared to healthy controls .In general, sensory adaptation and mental well-being scores were lower than other VF and QOL sub-scales Table\u00a0. AlthougP\u2009>\u20090.311, Fig.\u00a0A). However, anxiety was presented in 22 (23.7%) individuals in PSS group and in 10 (9.8%) individuals in healthy controls group. Depression was presented in 11 (11.8%) individuals in the PSS group and in 8 (7.8%) individuals in healthy controls group . The frequency of anxiety in PSS patients was significantly higher than that in healthy controls (P\u2009=\u20090.009). While the difference of the frequency of depression between the two groups was not statistically significant (P\u2009=\u20090.349).Although the mean scores for anxiety (6.98\u2009\u00b1\u20094.20) and depression (6.44\u2009\u00b1\u20093.66) in PSS patients were higher than the mean scores for anxiety (6.67\u2009\u00b1\u20093.21) and depression (5.96\u2009\u00b1\u20092.93) in healthy controls participants, no statistically significant difference was observed  but not with depression  using logistic regression for the whole participants.We found PSS was significantly associated with anxiety  and mental well-being score (P\u2009<\u20090.001) in QOL subgroup were significantly associated with anxiety. Mental well-being score (P\u2009=\u20090.004) was also significantly associated with depression. Anxiety and depression were not significantly associated with age, gender, smoking and drinking status, duration of disease, number of eyedrops used and other subgroups of VF and QOL scales.In univariate regression analysis in PSS patient group, mobility score (P\u2009<\u20090.001) and depression  after adjusted for variates with P\u2009<\u20090.2 in univariate regression analysis  , although depression was also more common  but statistically not significant. The present study also indicates that after adjusting potential confounding factors, mental well-being was significantly associated with anxiety and depression. Mental well-being was an independent risk factor of anxiety and depression.In this study, we assessed the anxiety and depression levels along with their potential risk factors in patients with PSS and healthy controls. Our results are important and are a value addition to existing literature and the association of chronic ocular diseases with psychological disorders. To the best of our knowledge, this is the first study to assess the anxiety and depression levels in patients with PSS and to determine the potential risk factors for these psychological conditions. Our results show the psychological vulnerability of patients with PSS. When compared with normal participants, PSS group had significantly higher frequency of anxiety  were greater than that in healthy controls (11.83% and 7.84%). Anxiety and depressive disorders are the two most prevalent mental disorders in the general population, with an estimated lifetime prevalence of 16% and 4\u201310%, respectively . AnxietyP\u2009=\u20090.044), which meant that PSS patients may think that others would be better off if they were not around, had lower spirits, and less confidence in their ability to perform. These negative emotions may contribute to the association between mental well-being and anxiety/depression. However, the association between anxiety/depression and chronic disease is not well understood. A systematic review reported that anxiety/depression could be the consequence of the diagnosis of a chronic disease, it could also be the cause of chronic disease, or the two conditions interact and exacerbated each other [We found mental well-being was an independent risk factor for both anxiety and depression. Mental well-being here is characterized as the feelings of being burden on others, dejection and loss of confidence . In thisch other . Our finWe did not find a correlation between age and anxiety/depression, which was consistent with a study by Lim et al. . In contThis study could be regarded as an initial exploration of the psychological state of PSS patients. However, there are also some limitations to the present study. First, there might be a potential selection bias as all the PSS patients were enrolled while they were visiting hospital, which meant that the disease was active. PSS patients in remission or those whose symptoms were cured were not included. However, in this study, we assessed the psychological status of patients with active PSS, who were affected more compared to others. Second, there might be some other factors related to clinical features or patient demography that could affect the psychological status of PSS patients that we did not analyse, such as IOP and visual acuity. Third, this was a cross-sectional study with limited sample size. Cohort studies with large sample size should be conducted to determine the relationship between chronic ocular diseases like PSS and their potential impact on the psychological status of the patient.In conclusion, our findings indicate that PSS patients may have higher anxiety and depression than healthy controls. Mental well-being was significantly associated with anxiety and depression in PSS patients and was an independent risk factor of anxiety and depression. Furthermore, it is important for ophthalmologists to be aware of the potential psychological comorbidities in PSS patients. This will help in disseminating appropriate information about PSS to prevent patients from developing undue anxiety/depression. Targeted interventions to improve mental wellbeing may also help in improving patient outcomes in patients with chronic ocular disease like PSS.Below is the link to the electronic supplementary material.Additional file 1 A copy of the HADS, VF and QOL questionnaire, along with the description of each sub-scaleSupplementary Material 1:"}
+{"text": "Interleukin-33 (IL-33), defined as \"alarming\", exert diverse functions through signaling via the suppression of tumorigenicity 2 (ST2). However, the physiological roles of IL-33/ST2 signaling during acetaminophen (APAP)-induced liver injury are still poorly understood by modern medicine (AILI). This research aims to explore the relationship between IL-33/ST2 and stimulator of interferon (IFN) response cGAMP interactor 1 (STING)-mediated signal transduction.C57BL/6N mice (WT) and IL-33-deficient mice (KO) were intraperitoneally injected with APAP (250\u00a0mg/kg). Recombinant IL-33 (500\u00a0ng/mouse) and the cGAS/STING inhibitor RU.521 (200\u00a0g/kg) were combined to treat AILI. For mechanistic research in vitro, CRISPR-mediated KD technology, immunoprecipitation, mass spectrometry, and immunofluorescence were utilized.We discovered that IL-33 deficient mice had increased APAP-induced hepatotoxicity, DNA accumulation, and type 1 IFN production. Mechanistic analysis revealed that IL-33/ST2 enhanced the interaction between Beclin-1 and STING, disrupting STING dimerization, IRF3 phosphorylation, nuclear transport, and IFN-1 gene transcription in HepaRG and Huh7 cells. Beclin-1 interacted with the C-terminus of STING, causing Lys338 acetylation and autophagy degradation of STING. ST2 depletion increased STING signal transduction and IFN-1 promoter activity. Surprisingly, the cGAS/STING inhibitor RU.521 and recombinant IL-33 together improved AILI in vivo.These results shed insight on the potential of inhibiting cGAS/STING as a therapy for AILI and emphasize the crucial role of IL-33/ST2 signaling in the regulation of APAP-induced STING signaling.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-023-01114-3. Acetaminophen , is an antipyretic and analgesic, which is mostly used worldwide as analgesics and antipyretics. However, APAP overdose gives rise to severe acute liver injury (ALI) and even acute liver failure (ALF) . It is wRecent studies have found that APAP-induced ALI is associated with the liver immune microenvironment. As an example, APAP overdose caused aberrant innate cell (Kupffer cells and neutrophils) infiltration and activation, and higher levels of pro-inflammatory TNF-\u03b1 and IL-6, but lower levels of anti-inflammatory IL-10 . IL-33, In the present study, we elucidate a novel insight that IL-33/ST2 signaling affected APAP-induced liver necrosis and type 1 IFN-mediated innate immune response during APAP-induced injury. Targeting STING signaling may hold further truthful research avenues for pharmacological interventions during drug-induced acute liver injury.Antibodies and reagents were obtained from the respective manufacturers: RP-MI-1640 medium , fetal bovine serum , penicillin\u2013streptomycin , Fluoromount with 4\u2019, 6-diamidino-2-phenylindole (DAPI) , Dulbecco\u2019s Modified Eagle Medium , Acetaminophen (APAP) , Lipofectamine 3000 , Recombinant human IL-33 and mouse IL-33 , puromycin (Solarbio), Sytox Green , anti-Flag , anti-Myc (ABMART), anti-HA (ABMART), anti-tubulin (ABMART), anti-LC3B , anti-Beclin-1 , anti-IRF3 , anti-p-IRF3 , anti-STING , anti-ST2 , anti-LaminB , Protein A/G PLUS Agarose (ABMART). cGAS/STING inhibitor RU.521 , 3-Methyladenine (3-MA) (CAS 5142\u201323-4), and MG132 (CAS 133407\u201382-6) were purchased from Merck, Darmstadt, Germany.Susumu Nakae . Our previous studies have verified the genotyping of mice [The male C57BL/6N mice (WT), weight approximately 18\u201322\u00a0g, six to eight-week-old, were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. The IL-33-deficient mice (KO) in the C57BL/N genetic background were kindly provided by Dr.  of mice . All mic of mice . In brie2 atmosphere.The HepaRG cells were obtained from Millipore  maintained in RPMI 1640. The HEK293T cells  and Huh7 cells  were maintained in Dulbecco\u2019s Modified Eagle Medium (DMEM). All cells were supplemented with 10% fetal bovine serum (Gibco) and 100 U/mL Penicillin/Streptomycin at 37\u00a0\u00b0C in a 5% COGutterman  which has been described elsewhere [1\u2212190, HA-STING191\u2212379, GFP-Golgi and Flag-Beclin-1 were obtained from Tsingke Biotechnology Co., Ltd. pCMV-C-HA, pCMV-C-Flag, pCMV-C-YFP, pCMV-C-Myc, pCMV-C-mCherry, and pCMV-C-EGFP were obtained from Beyotime. cDNA fragments prepared from HepaRG cells, amplified by PCR from cDNA, encoding human Beclin-1, ST2, STING, IRF3, TBK1 and cGAS were constructed and fused with Myc-, HA-, Flag-, YFP-, mCherry- or GFP, respectively, according to the manufacturer\u2019s instructions. The plasmids pCMV-HA-STING, pCMV-Flag-STING, pCMV-Myc-STING, pCMV-mCherry-STING, pCMV-HA-TBK1, pCMV-Flag-TBK1, pCMV-Myc-ST2, pCMV-Flag-ST2, pCMV-Flag-Beclin-1, pCMV-EGFP-Beclin-1, pCMV-YFP-IRF3, pCMV-HA-cGAS were constructed for this study. The primers used were given in Table SGFP-LC3 plasmid was kindly provided by Dr. lsewhere . HA-Ub, To establish HepaRG/shIL-33, HepaRG/shST2 and Huh7/shST2 cells, HepaRG and Huh7 cells were transfected with plasmids using Lipofectamine 3000 and selected by puromycin according to the manufacturer\u2019s instructions. To generate expression vectors pCDH-ST2 (ST2-OE), the coding sequences of the human ST2 gene were cloned into the NheI and BamHI sites of the control plasmids pCDH-CMV-MCS-EF1-copGFP vector . All constructs were verified by sequencing. The forward primer contained a NheI site 5\u2032-CTAGCTAGCATGATTGACAGACAGAGAATGGGAC-3\u2032, and the reverse primer the BamH1 site 5\u2032-CGGGATCCCCAAAGTGTTTCAGGTCTAAGCATGC-3\u2032. To build HepaRG/ST2-OE and Huh7/ST2-OE cells, HEK293T cells were co-transfected with the lentivirus expression vector  and pCDH-ST2 using Lipofectamine 3000. After 48\u00a0h, the lentivirus supernatant was harvested to infect HepaRG and Huh7 cells. Transduced HepaRG cells were selected by puromycin (3\u00a0\u03bcg/mL) for 3\u00a0days.Knockdown of the human ATG5 gene (ATG5 KD) using CRISPR/Cas9 technology. The guide RNA (gRNA) targeting the ATG5 was obtained from Tsingke Biotechnology. To generate the stable ATG5 knockdown cells, we ligated gRNA to the pX459 plasmid to obtain CRISPR/Cas9-gRNA plasmid, then the plasmid was transfected into HepaRG and Huh7 cells. The single-cloned cells were selected for expansion and culture. The efficiency of knockdown was confirmed by immunoblotting.The human IFN-\u03b21 promoter was obtained from the NCBI and cloned into the pGL4.10 vector . The sequences of the plasmids were verified using sequencing. HepaRG and Huh7 cells co-transfected with HA-tagged STING, pGL4-IFN-\u03b21 promoter-Luc reporter, or pGL4.10-basic control vectors for 24\u00a0h, were subjected to pre-incubation with or without rhIL-33 (100\u00a0ng/mL) for 2\u00a0h and then treated with 3-MA (10\u00a0mM) for 12\u00a0h, or proteasome inhibitor MG132 (10\u00a0\u03bcM) for 12\u00a0h. pRL-TK (Promega) was used as an internal control. Cell lysates were harvested for the dual-luciferase assay, which was performed according to the manufacturer's recommendations (Promega).In brief, cells were lysed in RIPA buffer  containing phenylmethylsulfonyl fluoride  and protease inhibitor cocktail  for 15\u00a0min at 4\u00a0\u00b0C. 5% of total lysates were used as input for each sample. The remaining lysate was incubated with 1\u00a0\u03bcg of primary antibody on the rotator at 4\u00a0\u00b0C overnight. Protein G sepharose was then added and incubated for another 4\u00a0h at 4\u00a0\u00b0C. Protein G sepharose-enriched complexes were resolved on SDS-PAGE gels and transferred onto PVDF membranes.Immunoblotting of the cell lysates and immunoprecipitates was performed using primary antibodies as indicated overnight at 4\u00a0\u00b0C and then with secondary antibodies for 1\u00a0h at room temperature. The bands were detected and visualized using a Hypersensitive ECL Chemiluminescence Kit .HA-tagged STING was transfected into HepaRG cells. At 24\u00a0h post-transfection, cells were treated with rhIL-33 (100\u00a0ng/mL) for 12\u00a0h. HA-STING protein precipitated by Beclin-1 was purified and then analyzed using mass spectrometry. Mass spectrometry was provided by Shanghai Luming Biological Technology co.Ltd.Nuclear and cytoplasmic extraction was conducted with the Nuclear and Cytoplasmic Protein Extraction Kit  according to the manufacturer\u2019s instructions. The extracted fractions were used for immunoblotting analysis.\u2212\u2206\u2206Ct was used to calculate the relative mRNA level. Data were normalized to GAPDH expression.Total RNA was extracted by Trizol reagent  following the manufacturer\u2019s instructions. Total RNA 2\u00a0\u00b5g) was used to perform reverse transcription with Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher). Quantitative real-time RT-PCR analysis was performed with a Quantstudio3 instrument (Thermo Fisher) using Fast Start Universal SYBR Green Mix . The primers, synthesized by Sangon Biotech., Shanghai, China., and used for real-time RT-PCR were displayed in Table S\u00a0\u00b5g was uSupernatants from mouse vessels were collected. Briefly, serum was separated by centrifugation at 4000\u00a0rpm for 10\u00a0min at various time points after APAP injection. Serum IL-33 levels were measured using commercially available ELISA kits (Elabsceience Bio.) following the manufacturer\u2019s instructions.p-value\u2009<\u20090.05 was considered statistically significant.All experiments were performed at least three times independently. GraphPad Prism 7 software  was used for statistical analyses. The results were presented as mean\u2009\u00b1\u2009SD. Statistically significant differences were determined by Student's unpaired t\u2011tests or one-way analysis of variance (ANOVA). Survival curves were compared using the log-rank (Mantel-Cox) test. A \u2212/\u2212 mice (KO). ELISA assay revealed a significant increase in serum IL-33 in WT mice after APAP administration at 6\u00a0h, 12\u00a0h and 24\u00a0h  for 12\u00a0h or a proteasomal inhibitor MG132 (10\u00a0\u03bcM) for 12\u00a0h. Immunoblotting analysis showed that ST2-mediated degradation of STING was completely recovered by adding the 3-MA but not MG132  truncation mutant or C-terminal (aa 191 to 379) truncation mutant to investigate the regions necessary for the Beclin-1 and STING interaction. In HepaRG cells, HA-STINGn-1 Fig.\u00a0A. Thus, n-1 Fig.\u00a0B. To tesNow that we have confirmed that IL-33/ST2 is associated with autophagy, what is the mechanism by which IL-33/ST2 affects the interaction of STING and Beclin-1? We purified the HA-STING protein precipitated by Beclin-1 from rhIL-33-stimulated cells and subjected it to LC\u2013MS/MS analysis. We identified a lysine peptide (HLRQEEKEEVTVGSLK) that was mapped to a region of STING containing K338 Fig.\u00a0D, indicaSTING is transported from the endoplasmic reticulum (ER) to the Golgi apparatus, recruiting TBK1 and then activating IRF3 . SubsequActivated STING recruitment TBK1 promotes the phosphorylation of STING, which in turn recruits IRF3 . PhosphoTo better verify the relationship between IL-33/ST2 and STING/TBK1/IRF3 signaling in the presence of APAP stimulation, the YFP-tagged IRF3 and HA-tagged TBK1 were co-transfected into HepaRG/shScram and HepaRG/shST2 cells treated with APAP. Confocal microscopy analysis suggested that the cytoplasmic IRF3 was reduced in HepaRG/shST2 cells. A similar trend was also seen in the presence of APAP stimulation Fig.\u00a0A. By extAs the knockdown of ST2 affected STING signal transduction, we further evaluated the transport and activation of STING in ST2-Vector and ST2-OE cells transfected with mCherry-STING upon APAP treatment. We found that the STING translocation from ER to a perinuclear compartment was increased in the presence of APAP stimulation. On the contrary, after the overexpression of ST2, this transformation was reversed Fig.\u00a0G. TogethHaving tested the important relationship between IL-33/ST2 and STING signal during APAP stimulation, we then sought to evaluate the therapeutic potential of cGAS/STING inhibition in APAP-induced liver injury. The cGAS/STING inhibitor RU.521 , 24 combAlthough APAP is a popular analgesic, an overdose results in severe centrilobular hepatocyte necrosis . The treA study revealed that they employed APAP at a dosage of 600\u00a0mg/kg, which is near the lethal dose of 750\u00a0mg/kg, and that this caused liver non-parenchymal cells to produce more DNA sensors than hepatocytes do during homeostasis . HoweverIL-33, a member of the IL-1 family of cytokines, defined as an \u201calarm protein\u201d released from necrotic cells, exerts diverse functions through signaling via its receptor ST2. Emerging studies have reported that IL-33/ST2 plays a critical role in the process of different liver diseases , 30. In Recent studies have shown that blocking STING/TBK1 signaling plays a central role in many diseases such as lung adenocarcinoma , chronicPost-translational modifications of STING proteins are critical for STING signaling. One study reported that phosphorylation at S366 of STING protein was required for IRF3 activation . HoweverMarques et al. provided that blockage of DNA recognition by the innate immune system might consist of a promising therapeutic avenue in DILI . TherefoIn conclusion, our findings established a pivotal role for IL-33/ST2 in regulating the activation of cGAS/STING in APAP-induced liver injury. Targeting STING signaling may be a novel strategy for the treatment of acetaminophen hepatotoxicity.Additional file 1.Additional file 2: Table S1. The primer sequences used for cDNA amplification.Additional file 3:Table S2. The primer sequences used for cDNA real-time RT-PCR."
\ No newline at end of file