To determine the frequency of FTs genome-wide, we developed a TopHat-Fusion-based analytical pipeline to identify FTs with high specificity. We applied it to an RNA-seq dataset from the nucleus accumbens (NAc) of mice repeatedly exposed to cocaine. Cocaine was previously shown to increase the expression of certain REs in this brain region. Using this pipeline that can be applied to single- or paired-end reads, we identified 438 genes expressing 813 different FTs in the NAc. Although all types of studied repeats were present in FTs, simple sequence repeats were underrepresented. Most importantly, reverse-transcription and quantitative PCR validated the expression of selected FTs in an independent cohort of animals, which also revealed that some FTs are the prominent isoforms expressed in the NAc by some genes. In other RNA-seq datasets, developmental expression as well as tissue specificity of some FTs differed from their corresponding non-fusion counterparts. Finally, in silico analysis predicted changes in the structure of proteins encoded by some FTs, potentially resulting in gain or loss of function. Collectively, these results indicate the robustness of our pipeline in detecting these new isoforms of genes, which we believe provides a valuable tool to aid in better understanding the broad role of REs in mammalian cellular biology.Repetitive elements (REs) comprise 40–60% of the mammalian genome and have been shown to epigenetically influence the expression of genes through the formation of fusion transcript (FTs). We previously showed that an intracisternal A particle forms an FT with the  Epigenetic modifications such as DNA methylation and post-translational changes of histone tails have been shown to influence the expression of protein coding regions across the genome. Interestingly, the approximately 5 million repetitive element (RE) loci, which collectively comprise 40–60% of the mammalian genome  are alsoAvyagouti mouse mutant showed that an intracisternal A particle (IAP) upstream of the first coding exon of agouti, when epigenetically unsilenced, hijacked expression of the gene [agouti. Transcription from the IAP led to expression of the agouti protein ubiquitously throughout the animal, and resulted in a phenotype involving yellow fur, obesity and the development of type 2 diabetes by 8 weeks of age [Avy trait was later shown to be strongly influenced by the environment: a variety of compounds in the mother’s diet change the penetrance of Avy in the offspring by affecting the methylation status of the IAP [While the full extent of the biological impact of expressing REs is still poorly understood, one known outcome is the ectopic regulation of adjacent protein-coding genes through the formation of FTs. For instance, earlier work involving the analysis of three independent alleles of the viable yellow the gene ,7. This s of age ,7. It was of age ,7. The p the IAP .agouti was one of the first examples, the use of next generation (NextGen) sequencing tools has more recently revealed broad-based formation of FTs across the mammalian genome involving different types of repeats and genes [Although nd genes ,5,11–16.nd genes . ThorougTo gain more insights into the frequency, abundance and expression pattern of FTs, here we describe the development of a sensitive and highly specific bioinformatics pipeline applied for the genome-wide detection of FTs using RNA-seq datasets. We applied this pipeline to the analysis of data generated from the nucleus accumbens (NAc), a key reward structure of the brain, of cocaine-treated mice ,19. Repead libitum. Animals were killed by decapitation without anesthesia or analgesia, since all known anesthetic and analgesic agents affect the neural circuits and their biochemical constituents that are under study. Such decapitation occurs within a second and is standard practice. The health of all animals is monitored at least once daily. All animal protocols were approved by the Institutional Animal Care and Use Committee of Mount Sinai. Animals received daily i.p. injections for 7 consecutive days of cocaine HCl (Sigma) at 20 mg/kg body weight. Mice were used 24 hr after the final injection. Control mice for all groups received saline injections. Bilateral 14-gauge NAc punches were taken from each animal.Adult male C57BL/6J mice (Jackson laboratory) approximately 8 weeks old were used in this study. Animals were habituated for at least 1 week before experimentation. They were housed 5 per cage on a 12-hr light-dark cycle at constant temperature (23°C) with access to food and water RNA isolation and RNA-seq were performed as described previously . In briewangt4@niehs.nih.gov).Fusion-detecting tool TopHat-Fusion version 2.0.4 was used for identification of FTs for each sample . The folhttp://www.sanger.ac.uk/resources/mouse/genomes/), as well as from different developmental stages including the oocyte and 2 cell-stage [Additional publicly available RNA-seq data sets were used to determine the tissue- and stage-specificity of FT expression. They are from various brain regions , from other tissue types . Gene annotations were based on KnownGene, which was also downloaded from the UCSC Genome Browser. To identify the chimeric reads, the GenomicRanges package of R/BioConductor version 2.14 was used. Reads that did not map full-length to the genome and had 25 bases at one end derived from a repeat locus (not mapping within a defined exon) and the other 25 bases to an exon were considered fusion reads. Scripts were developed to check the uniqueness of the 25 bases aligning to the exon, which was then used to define the identity of the candidate fusion gene which was recorded. The estimated pool of candidate genes that express FTs was calculated based on the identified genes relative to the total number of annotated genes (~29K) on mm9.Short read aligner Bowtie version 1.0.0 with minor modifications on the default settings (-v 1 and -m100K) was used to estimate the number of chimeric reads or fusion genes in the NAc transcriptome. REs were defined by RepeatMasker and downloaded from the University of California Santa Cruz (UCSC) Genome Browser  and their nearest REs using the gene model (.gff3) and RepeatMasker annotations from mm9. The frequencies were calculated per RE class  based on the genomic region of the repeat (intronic or intergenic) from which start and end positions were recorded. Exons were used as anchors to define the nearest repeat, and the intronic or intergenic frequencies of each RE class summed. As the start and end positions of REs were recorded, statistics were run separately to confirm each other. The absolute frequency for each RE class is shown as the sum of these values per genome region, which as expected, was comparable. The script is available upon request to Dr. Gonzalo Riadi .For biological replication, a separate cohort of animals was prepared for cocaine i.p. injection and tissue collection. After isolation via RNeasy Micro columns (Qiagen), RNA was then reverse transcribed using a Bio-Rad iScript Kit. cDNA was quantified by quantitative PCR (qPCR) using SYBR Green (Quanta). Each reaction was run in duplicate or triplicate and analyzed following the ΔΔCt method as previously described using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or beta-2-microglobulin (B2M), which are not altered by cocaine exposure, as normalization control. The PCR products were verified on an agarose gel and Sanger sequencing was used to confirm the size and sequence accuracy. For comparisons of the levels of expression of non-fusion vs FT isoforms, primers were designed such as to amplify the RE and exon present in the FT, and the same exon but when spliced to the next non-fusion control locus, as shown in As presented on p-value for a FT was determined as the number of times (n) that a test statistic score from the comparisons of the bootstrapped samples was > the positive observed test statistic score or < the negative observed test statistic score. Hence, p-value = n/B; the p-value of each event was calculated using R package version 2.14.1.Additionally, for each FT, we applied the Wilcoxon sum of ranks test to the raw RNA-seq fusion counts comparing read counts from saline to cocaine for each event to obtain an observed test statistic score. To increase confidence of those findings, we also performed bootstrapping sampling  with replacement. In brief, based on read counts from within each group we randomly sampled combinations of read counts 10,000X, which were then used to compare each fusion event between saline and cocaine. This generated a non-parametric distribution of test statistic scores. The To identify FTs with high specificity, we developed a bioinformatics pipeline centered around the software package TopHat-Fusion, which was created to identify chimeric transcripts arising from chromosomal translocations in cancer . We reasWe studied all 4.9 million repeat loci including the most common classes of REs present on the mouse genome assembly (mm9): Long Interspersed Nuclear Elements (LINEs), Short Interspersed Nuclear Elements (SINEs), LTRs, DNA repeats and satellites. A summarized analytical workflow is shown in Read counts spanning the junction site of the fusions varied from 1–3 to 100s, depending on the gene . BecauseDrd2) FT was not statistically different based on read counts but proved to be so by qRT-PCR  the data. Bootstrap essentially repeatedly  reassigns random values generated from the read count distribution within a group, populating a distribution of gene specific scores that overall increases the resolution of the test  and Celf2  were expressed at 40- and 10-fold higher levels, respectively, than their non-FT counterparts (Pgc1α), FTs were present at about equal levels to the corresponding non-FT isoform. In other instances (see Arhgef10 (Rho guanine nucleotide exchange factor 10), where the fusion A and B transcripts were expressed at levels that were less than 5% of the non-FTs from the gene.Having confirmed the specificity of the TopHat-Fusion pipeline in detecting these new gene isoforms, we next asked how the level of expression of the FT related to the expression of the non-fusion counterpart. To address this we performed qRT-PCR on 8 of the 20-PCR validated genes, which were prioritized based on counts and responsiveness to cocaine . We usedterparts . For thences see , FTs werAtp2b1, Celf2, Klc1 (Kinesin light chain 1) and Srrm2 (serine/arginine repetitive matrix 2) revealed that FTs from these genes were expressed in all tissues analyzed  and Elfn2 (extracellular leucine-rich repeat and fibronectin type III domain containing 2) were clearly tissue specific with FTs found in the brain but not in any other tissue analyzed  and publicly available datasets from various tissues  as well as from different developmental stages (see below). We compared the number of reads that mapped over the junction of the repeat-exon (FT counts) to the reads that mapped over the corresponding exon-exon junction of the same gene (non-FT counts). Analysis of analyzed ; levels  studied . Conversanalyzed .Atp2b1, Celf2 and Srrm2. Their expression differed significantly from the expression of their corresponding non-FT isoforms  and decreased ~5-fold in the adult; expression of the non-fusion isoform of Klc1 only became apparent at later stages . The non-FT isoform generally appeared to be more abundantly expressed at early developmental stages , the granular layer (L4) and the superficial L2/3 or supragranular layers (SgLs) . We anallt brain . The revr stages . For Cell stages . The patWe next asked about the impact of the FT to either gene expression regulation or to protein structure. To this end, we used the sequence of the fusion reads to reconstruct the genomic configurations that likely give rise to the FTs. We used the annotated intron-exon (KnownGene) and repeat coordinates on mm9 of 27 selected genes that produced at least one FT. From these analyses we determined not only that all of the different classes of REs studied can generate FTs but also that REs can function as alternative promoters, become new exons and/or lead to truncation of proteins. The genomic configuration of three genes giving rise to FTs illustrating the different ways that a RE can influence the expression of the gene through the formation of FTs is shown in Atp2b1 (panel A). Atp2b1 is a gene that is functional in a broad spectrum of cell types. The FT from Atp2b1 involves a SSR 359 bases upstream from the annotated transcriptional start site (TSS). The SSR appears to function as an alternative promoter, forming a new transcript that splices to the first coding exon of the gene containing the annotated translation start site, which is about 54Kb away. Our ChIP-seq analysis of the NAc from saline- and cocaine-treated animals [agouti [Atp2b1 involves a RE likely functioning as an alternative promoter that regulates expression of a wild-type protein. Inspection of available mouse ESTs (expressed-sequence tags) at the National Center for Biotechnology Information (NCBI) confirm the presence of isoforms containing a fragment that maps to the SSR coordinates.The first gene in  animals  revealed [agouti ,7, the FPgc1α. Pgc1α derives its name from its interaction with the peroxisome proliferator-activated receptor gamma (PPARγ) and co-activates specific downstream targets of this nuclear receptor within the cell. The FT that we first identified for this gene involves a portion of a SINE, located about 365Kb upstream of the annotated promoter that is spliced to the second coding exon , but, most notably, to potentially alter the structure of the protein being produced from that gene by either adding amino acids or skipping exons.Overall, reconstructing the genomic configurations of these 27 genes revealed that FTs have the potential to not only provide alternative promoters  across mm9, the computational demands of the software proved beyond the memory capacity of our servers (~4TB). Additionally, our requirement that the RE be spliced to a protein coding exon, which was necessary to exclude reads derived from primary unspliced transcripts , unfortunately also filtered out reads from FTs arising as contiguous fusion events with the adjacent genomic sequences, similar to the transcripts from the Given these limitations, we sought an alternative approach using the software program Bowtie to better estimate the number of genes that may give rise to FTs across the genome. This software is not designed to detect FTs but is computationally efficient to map reads multiple times. The concept underlying this approach was to focus on reads that did not map full length, as they would likely be chimeras, and hard clip 25 nucleotides from each end of the read. We would then map each clipped section independently to mm9, and analyze those reads in which one end mapped to a RE while the other mapped to a protein coding exon. By anchoring the analysis on the exons we could then identify from which gene the fusion candidate was derived.Implementation of the hard clipping analysis started by mapping reads from our saline and cocaine RNA-seq datasets to up to 100K loci (-m100K) to mm9. From the initial pool of ~96M reads in each dataset, we found that ~25M reads did not fully map to the reference genome . Next, wArhgef10, Drd2 and Pgc1α indicated that hard clipping identified the same events. Additionally, using the chromosomal coordinates of the 25 bases aligning to the RE and the exon we reconstructed 3 events only detected by hard clipping that involved repeats mapping more than once. We found that while repeats mapped to several locations, including in different chromosomes, in all cases there was at least one RE on the same chromosome and in close proximity (up to 5Kb) to the identified exon (data not shown). While not comprehensive, we conclude with these limited analyses that the hard clipping strategy may be informative in estimating broadly the number of potential FT-expressing genes. Currently, this number suggests that ~18% of genes across the mouse genome are likely to express FTs.This simple strategy utilizing hard clipping identified an average of 5,553 candidate genes expressing FTs from either the cocaine or saline groups . About 8It is becoming increasingly clear that REs can have a substantial impact on the transcriptome of mammalian cells. In fact, most recently different analytical pipelines have been developed to analyze RE expression –29. HowePrevious work has demonstrated that epigenetically activated REs have two major biological outcomes. The first is the ability to retrotranspose, for those elements that have retained this capacity such as LINEs and SINEs, which may ultimately cause new insertional mutations. While this effect can be detrimental, as in cancer , others Pgc1α fusion is ~565Kb away from the annotated TSS of the gene, and the SSRs involved in the Celf2 fusions are not only intronic but about 13Kb away from the closest annotated TSS , our data indicate that this may not always be the case. For instance, the SSR from the Atp2b1 and Arhgef10). They can also be included as new coding exons that may change protein sequence and presumably function (e.g. Pgc1α and Klc1) and, most interestingly, potentially give rise to truncated proteins (Arhgef10 fusion B). We also predict that subcellular localization of proteins translated from FTs may be changed, with the repeat-fusion creating or disrupting the targeting signal of an isoform . This may be a previously unappreciated way to increase the repertoire of isoforms of a gene/protein in the cell. However, it has yet to be determined that any of the FTs expressed in our data set gives rise to such proteins. Of note, a SINE-containing human brain-specific isoform of Pgc1α, which initiates transcription in a novel non-annotated upstream alternative promoter, has recently been reported and shown to be translated into protein [Our data also reveal that there are several mechanisms through which REs can influence the expression of a gene. As previously shown by us and others ,6,16, we protein .Finally, using different statistical methods we identified FTs in which read counts were significantly different between saline- and cocaine-treated animals. However, qRT-PCR failed to validate some of the differential expression that was found to be statistically significant. In other cases, we found that qRT-PCR revealed differential expression that was not statistically significant by counting RNA-seq fusion reads. Some of this lack of reproducibility may be attributable to the natural biological variation between the two independent sets of animals used for the two different approaches. Another possibility is that the read counts for the fusion reads, which are only considered if they cross the exact fusion junction, may not adequately reflect the true level of expression of the entire FT. Until a better means of determining FT expression level is developed, alternative methods will be necessary to more accurately test differential expression of FTs in response to, for instance, an environmental exposure such as cocaine.In conclusion, we developed a highly specific pipeline to detect FTs genome-wide using data resulting from widely available RNA-seq technology. While we based our analysis on the TopHat-Fusion platform, many fusion transcript detection software packages are available and could be the basis for similar analysis . In factS1 FigTopHat-Fusion was used to identify FTs. Reads were allowed to be mapped to one unique genomic locus on the reference genome to increase specificity of findings. The stringent criteria adopted to determine whether a transcript was expressed as a fusion are depicted in the figure.(PDF)Click here for additional data file.S2 FigArhgef10 fusion A (lane 1) and fusion B (lane 2) are depicted as representative of the fusion events validated. Gel has been cropped to remove irrelevant data; DNA marker was present in the same gel. Sequence of primers used for the PCR can be found in Reverse-transcription PCR was performed to determine expression of FTs in RNA samples from the NAc of animals exposed to saline or cocaine. (PDF)Click here for additional data file.S3 FigRead counts were compared between FTs and non-fusion isoforms in different areas of the brain including the amigdala (AMY), hippocampus (HIP), NAc, prefrontal cortex (PFC) and the ventral tegmental area (VTA) as well as in different tissues. Black bars represent average counts from 4–6 independent samples of the fusion version of a transcript while white bars indicate the average for the non-fusion counterpart.(PDF)Click here for additional data file.S4 FigPattern of expression of fusion or non-fusion isoforms as a function of neocortex development. Bars represent average read counts ± SE from 2 samples ; thus statistical significance could not be tested. Data from 3 different layers were analyzed: IgL (infragranular layer), L4 (granular layer) and SgL (supragranular layer). P4–14 represent post-natal days 4 to 14.(PDF)Click here for additional data file.S5 FigRepresentation of the fusion events predicted in 24 additional randomly selected genes out of the 813 events detected. The canonical start codon (ATG) is shown based on gene annotation; directionally of transcription is indicated with red arrow above and below the panels based on UCSC gene annotation. In all cases, top panel shows full view of gene structure. TSS indicates the transcription start site. Middle and/or lower panels show enlarged view of different predicted fusions, including the splicing recognition sites (GT-AG). Orange or green brackets indicate regions involved in the fusion (repeat/exon or vice-versa). Red boxes indicate the region from which RNA-seq reads support the fusion events; when more than one fusion was identified on the same gene (isoform) green boxes identify the reads supporting the additional event. The number of reads supporting the fusion event is also depicted.(PDF)Click here for additional data file.S6 FigChIP-seq was performed using different histone marks and RNA polymerase II (for more details see reference (PDF)Click here for additional data file.S7 FigRepeatMasker track indicate in grey the SSR involved in this fusion. Below the nucleotide sequence of the repeat in various organisms is shown. The chromosomal coordinates for the SSR is shown above the figure.(PDF)Click here for additional data file.S8 FigRepeatMasker track indicate in grey the SINE (upper panel) and the SSR (lower panel) involved in this fusion. The nucleotide sequence of the repeat in various organisms is shown below the RepeatMasker track; the genomic coordinates of each individual repeat are shown above each panel on the figure.(PDF)Click here for additional data file.S1 TableGene name, fusion type, chromosomal coordinates, direction of the fusion and read count in saline- or cocaine-treated samples are shown. The type of repeat, the family and class of the RE as well as the strand of the elements involved in the fusion are also depicted. S1–3 reflect each of the cocaine samples  and S4–6 refer to saline-treated controls . Statistical analysis was performed based on raw read counts as well as fold-change using Wilcoxon test. In bold are the p-values that are statistically significant with p≤0.05.(XLSX)Click here for additional data file.S2 TableForward (F) and reverse (R) primers were designed to amplify the RE and exon present in each FT. In some cases the same exon was amplified with a primer from an adjacent exon in order to compare the level of the FT with that of the non-fusion isoform .(XLSX)Click here for additional data file.S3 TableThe list of 813 FTs from  was used(XLSX)Click here for additional data file.S4 TableOnly gene name is shown. In bold are the genes also identified by the TopHat-Fusion pipeline as FT-expressing genes.(XLSX)Click here for additional data file.


Here we report on the resulting amino acids found using the Titan post-impact conditions in a classical Miller experimental reaction set-up and how they differ from the simulated early Earth conditions.The problem of how life began can be considered as a matter of basic chemistry. How did the molecules of life arise from non-biological chemistry? Stanley Miller’s famous experiment in 1953, in which he produced amino acids under simulated early Earth conditions, was a huge leap forward in our understanding of this problem. Our research first simulated early Earth conditions based on Miller’s experiment and we then repeated the experiment using Titan post-impact conditions. We simulated conditions that could have existed on Titan after an asteroid strike. Specifically, we simulated conditions after a potential strike in the subpolar regions of Titan that exhibit vast methane-ethane lakes. If the asteroid or comet was of sufficient size, it would also puncture the icy crust and bring up some of the subsurface liquid ammonia-water mixture. Since, O’Brian, Lorenz and Lunine showed that a liquid water-ammonia body could exist between about 10 His expnditions . In eachOur research first simulated early Earth conditions based on Miller’s experiment and then reproduced it using conditions that may occur on Saturn’s largest moon Titan. Specifically we modeled conditions that could occur after a large impact on Titan. We simulated conditions that could have existed on Titan after an asteroid strike . Specifi2O and boiled for 24 h to thoroughly flush the system. The water was then removed and another rinse with 200 mL of ddH2O was completed. An apparatus was built replicating the original apparatus used by Stanley Miller in 1953 [2O was added back to the round bottom flask. Vacuum was applied for 10 min to purge the solution of gases. Hydrogen gas was then added to a pressure of 1 bar followed by application of vacuum for another 10 min. The vacuum purge and hydrogen flush procedure was repeated 3 times. After the final vacuum purge, gases were added in the following order to the following pressures: (1) hydrogen gas, 0.2–0.3 bar, (2) methane gas, 0.2–0.3 bar and (3) ammonia gas 0.2–0.3 bar. The total pressure inside the apparatus approached 1 bar. All stopcocks were then closed and a cooling water bath was applied to the condensation tube. Heat was applied using a Staco variable transformer and the solution was brought to a constant boil. After a constant boil was achieved the Tesla coil attached to a manual timer was used to apply a spark  to the electrode in the upper chamber (After the cleaning procedures were completed approximately 200 mL of ddH chamber b. The sp chamber a. These The round bottom flask was filled with 200 mL of 30% aqueous ammonia and the apparatus was placed under vacuum for 10 min to purge the solution of gases. The system was then flushed with hydrogen gas and placed under vacuum three times (10 min each time) to completely purge the system and solution. Gases were then added in the following order to the following pressures: hydrogen gas to 0.05 bar, methane gas to 1.0 bar and ammonia to 0.45 bar . The water was then brought to a boil and the spark was applied using the Tesla coil up to a maximum of 45,000 volts b. The spAll samples were run on a Waters MALDI Q-TOF Premier Micromass instrument in ESI+ Q-TOF W mode using direct injection. The injection method was direct using a 1 mL GC syringe coupled to a small motor so that the sample was injected continuously over a period of 10 min. All samples were diluted 1:10 in water before injection. A solution of 10% formic acid/0.1M sodium hydroxide/acetonitrile at a ratio of 1/1/8 was used to calibrate the instrument. The peaks from each sample with an intensity value above 15% were again run in MS-MS mode to help with identification and analysis. Post-run processing of samples was done on the MassLynx software from Waters Laboratories Informatics and included peak smoothing using the Savitzky Golay method and peak centering. Both processes reduced the background and limited peak widths to a value of 6 at half height.The peak lists were then annotated based on the relative intensity of the peak. For example, in Titan time course sample 1 only peaks with relative intensities above 3 were considered. In other samples the background was such that only peaks with relative intensities above the background noise were considered. For example, in Titan time course sample 2, the background was such that only peaks with relative intensities over 40 were considered in the analysis.After each file was annotated, the metabolite MS and MS-MS database from the Scripps Center for Metabolomics was used to search for peak matches . The cutThe amino acids observed from the early Earth conditions are very similar to the amino acids seen in the original Miller experiment and in several follow-up experiments ,9. In StThe Titan time course experiments produced a range of amino acids as seen in The starting solution of 30% aqueous ammonia is shown in the representative spectrum in m/z peaks on the spectra represented multiple ring-type structures and are mostly occluded by the higher background.These samples were taken 2 days after the experiment was started when we noticed a pink-red clear liquid pooling near the vacuum stopcock joint area prior to the atmospheric chamber. The joint area is located at the stopcock that leads to the inlet port for the gases used in the experiment. The amino acids in this sample are found in complexes of two to three amino acids (mostly groups of two). Specifically, glutamine, alanine, tyrosine, serine and phenylalanine were found in groups of two to three while glutamic acid, arginine, leucine, isoleucine and valine were found in separate groups of two to three amino acids each. The higher m/z peaks contained multiple ring-type structures and were mostly occluded by the higher background.These samples were taken 4 days after the experiment started and had a slight pink color but had not reached the bright pink color seen at the end of 7 days. Samples were collected from the sample stopcock at the bottom of the apparatus. The amino acids were found in complexes of two to three amino acids. In this sample, arginine, proline, serine, aspartic acid, leucine, valine, isoleucine, glutamic acid, tryptophan, phenylalanine and histidine were seen in groups of two to three amino acids each. The most amino acids were identified from sample 3 as well as the most number of two to three amino acids groupings. The higher These samples were taken at the end of the 7 day experiments from the 250 mL round bottom flask using the sample stopcock. The samples were all a light pink in color. In sample 4 no amino acids were identified but several large ring structures were present. These structures included proanthocyanidin, alpha-L-rhamnopyranosyl-(1->2)-beta-D-galactopyranosyl-(1->2)-betva-D-glucuronopyranoside and a diethylstilbestrol diphosphate-like compound. All of these compounds have two or more benzene-like ring structures. The identification of a diphosphate-like compound was of some concern since no phosphorus was added to the reaction mixture. However, this could be explained by some carry-over from the cleaning solutions used on the Miller-Urey apparatus or as a lack in the database of spectra. Since the diphosphate compound didn’t appear on the spectra for samples 2 and 3, and since it did not appear in the starting solution, the problem is most likely one with the database.We were successful in replicating the original Miller-Urey early Earth conditions and produced many of the same amino acids seen by Stanley Miller. In particular we identified alanine, glycine and aspartic acid just as Miller did. Our experiments also identified several more amino acids than Miller , which cIn contrast, in the Titan time course samples we see an intriguing pattern emerge. Amino acids start to be produced 2 days after the application of spark and continue to increase in structural complexity (groups of two amino acids and then groups of three amino acids) up to 4 days after the spark is applied. Samples taken 2 days after the start of the experiment were taken from the vacuum stopcock because this is where we noticed a distinct pink-red clear solution. This pink color could be due to organonitrogen, like the pink-orange haze on Titan that is due to organic photochemistry in the atmosphere . This piBoth the 2 day and 4 day time points included the amino acids serine, phenylalanine, glutamic acid, arginine, leucine, isoleucine and valine. While glutamine, alanine and tyrosine were observed in the 2 day sample, they were not present in the day 4 sample. However, at 4 days post-spark application we did observe histidine, aspartic acid, proline and tryptophan. The emergence of aspartic acid and the absence of alanine in the 4 days sample could be attributed to the pH of the sample mixture. In previous spark discharge experiments it has been noted that acidification of the reaction mixture reduced the yield of amino acids observed at the end of the experiment . In our et al. [While our experiments are the first to simulate Titan post-impact conditions, other researchers have completed simulations of Titan atmospheric conditions. Due to the presence of large, complex organic molecules in Titan’s upper atmosphere, Horst et al.  simulateet al. . The onlOverall, our experiments show that it is possible to produce a wide range of amino acids on Titan in post-impact conditions. This finding could have important implications for the chemical evolution and possibly for an origin of life on Titan, either in the past or even persisting to the present day. It is especially interesting that we observed a wider range of amino acids in the Titan condition experiments than in the early Earth condition experiments. These amino acids were also always found in groups of two three and not individually. Even the eventual break down of amino acids observed at the 7 day time point could have implications for life on Titan since the larger carbon ring structures could also be important in the chemical “soup” of life by acting as scaffolds for the building of chains of amino acids. On the other hand they could, however, be just a tar-like substance presenting a dead-end scenario for the development toward further complexity. If the latter scenario is correct, it would mean that the availability of amino acids for further organic synthesis reactions may be severely time-constrained and reactions toward higher prebiological complexity have to set in within a few days after the impact event in order to proceed. The observation that these amino acids are grouped together may provide a hint that such reactions are indeed occurring. Further experiments and analysis of the created organic compounds are needed to resolve this important question.The Titan post-impact reaction conditions produced amino acids beginning at least 2 days post application of spark and continued to increase in quantity and complexity (groupings of two to three amino acids were observed) up to 4 days after the spark was applied. The most common amino acids observed were serine, phenylalanine, glutamic acid, arganine, leucine, isoleucine and valine. However, after 7 days of experimental activity the groupings of amino acids seen at day 4 started to break down and form large carbon ring structures. Our results show that not only is it possible to produce amino acids under Titan post-impact conditions, we see different amino acids from those identified in the original Miller conditions. Further experiments using our Miller apparatus will include the addition of sulfur to the reaction mixture as well as trace gases (tholins) found in the atmosphere of Titan.


This study aims to assess the most efficient combinations of vaccination and screening coverage for the prevention of cervical cancer (CC) at different levels of expenditure in Nigeria.An optimization procedure, using a linear programming approach and requiring the use of two models , was developed. The evaluation model, a Markov model, estimated the annual number of CC cases at steady state in a population of 100,000 women for four alternative strategies: screening only; vaccination only; screening and vaccination; and no prevention. The results of the Markov model for each scenario were used as inputs to the optimization model determining the optimal proportion of the population to receive screening and/or vaccination under different scenarios. The scenarios varied by available budget, maximum screening and vaccination coverage, and overall reachable population.In the base-case optimization model analyses, with a coverage constraint of 20% for one lifetime screening, 95% for vaccination and a budget constraint of $1 per woman per year to minimize CC incidence, the optimal mix of prevention strategies would result in a reduction of CC incidence of 31% (3-dose vaccination available) or 46% (2-dose vaccination available) compared with CC incidence pre-vaccination. With a 3-dose vaccination schedule, the optimal combination of the different strategies across the population would be 20% screening alone, 39% vaccination alone and 41% with no prevention, while with a 2-dose vaccination schedule the optimal combination would be 71% vaccination alone, and 29% with no prevention. Sensitivity analyses indicated that the results are sensitive to the constraints included in the optimization model as well as the cervical intraepithelial neoplasia (CIN) and CC treatment cost.The results of the optimization model indicate that, in Nigeria, the most efficient allocation of a limited budget would be to invest in both vaccination and screening with a 3-dose vaccination schedule, and in vaccination alone before implementing a screening program with a 2-dose vaccination schedule. The incidence of cervical cancer (CC) in the Sub-Saharan Africa region, where Nigeria is located, is among the highest in the world. The CC incidence per 100,000 in Sub-Saharan Africa is 19.1[In many developed countries, where national routine screening programs using the Papanicolaou (Pap) smear have been implemented, the CC incidence and mortality have been significantly reduced-5. EarlyVaccination provides an alternative or a supplementary intervention for CC prevention. Infection with human papillomavirus (HPV) has been shown to be a necessary condition for the development of CC-10. EighNumerous studies have investigated the cost-effectiveness of HPV vaccination or CC screening in many countries in Europe, Africa, and Latin America, and most have concluded that both methods of prevention are cost-effective.Standard cost-effectiveness or budget-impact analyses are however not the best methods to determine which mix of prevention strategies provides the most efficient use of limited resources. Standard cost-effectiveness analyses do not typically take into account affordability constraints when estimating the cost-effectiveness of different combinations of prevention strategies, and are also limited in their ability to examine the comparative efficiency of many different combinations of prevention interventions. Because neither vaccination nor screening alone can provide 100% protection against CC, an optimal prevention strategy might include a combination of both. Budget-impact analyses typically estimate only affordability and do not link budget impact to health outcomes.An alternative approach to economic assessment is optimization modeling applied previously in many different areas such as transport, agriculture, industry, and banking, and morOptimization modeling provides valuable additional information compared with either cost-effectiveness or budget-impact analysis, since it explicitly evaluates multiple available options to select the combination that fulfills all the constraints introduced in the model while obtaining the most efficient result: lowest cost for the best outcome-28. ThisThe goal of this analysis was to provide information for Nigeria, as an example of a Sub-Saharan African country currently investigating a solution to improve CC prevention, about the most efficient combinations of prevention and screening coverage at different levels of expenditure. Nigeria has a population of about 170 million and is also the most populous country in Africa with a high CC burden. MoreoveWe used an optimization model to identify the combinations of vaccination and screening coverage that would provide the greatest estimated reduction in the annual CC incidence for different levels of expenditure per woman in Nigeria. This information can be used by policy-makers in Nigeria and other countries in Africa with similar CC incidence and mortality rates when designing strategies to reduce the CC burden in their country.The optimization model used in this study to identify the optimal combination of CC prevention strategies in Nigeria has been applied previously to the United Kingdom (UK) and Brazil to run similar analyses. This evThe optimization procedure requires the use of two models. The first model embedded within the optimization procedure was a Markov cohort model: the “evaluation” model. It was used to generate estimates of the annual incident CC cases at steady state in a population of 100,000 women for each of four alternative strategies considered in the evaluation: screening only; vaccination only; screening plus vaccination; and no prevention. The number of incident CC cases was chosen as the primary outcome measure because CC prevention represents the ultimate goal of screening or vaccination. The results of the Markov model for each scenario were used as inputs to the optimization model. The optimization model was then used to determine the optimal mix of interventions for maximizing the reduction in CC incidence under different scenarios. Those scenarios varied by available budget and by constraints on the maximum screening and vaccination coverage to be reached and the overall reachable population. Alternative scenarios were considered by varying the screening and vaccination coverage constraints to model different feasible intervention uptakes within a Sub-Saharan African country such as Nigeria. The purpose of testing different budget scenarios was to reflect the reality of limited health care funding and to demonstrate the incremental reduction in CC cases that would be possible with additional spending. This evaluation is intended to inform decision-makers about the health and economic impact of different prevention strategies as well as the optimal potential program.Excel was adapted to the Nigerian setting and was used to estimate the clinical and cost outcomes associated with each specified prevention strategy analyzed separately among a female population[A previously developed Markov cohort model built in Microsoft pulation-33. ScreThe natural history transition rates between the different model health states were assumed to be the same as those used in the original Markov model and wereThe validity of the model was assessed by comparing the estimated age-dependent CC incidence without any prevention strategy to the CC incidence reported in GLOBOCAN. CalibraHealth care services used for treating CIN grade 1, CIN grades 2 and 3, and CC were taken from a retrospective chart review performed at the university college hospital at Ibadan where patients’ charts are archived. The chart review collected the medical resources used  to treat a patient with CIN1, CIN2/3 or CC. This study received approval from the University of Ibadan/University College Hospital Ethics Committee. For precancerous lesions, resources used over a 1-year period from the date of diagnosis were collected; for CC, lifetime resources were collected (from diagnosis until either death or cure). For each condition, 10 patients with the required information at the time of data collection (2010) were identified  and the medical resources used were extracted from their hospital medical records. The associated costs were estimated after assigning unit costs from the hospital record to each of the medical resources used. The costs were adjusted to 2012 values based on the Nigerian consumer price index for the health care sector. The aveVaccine efficacy was estimated as the weighted average vaccine efficacy of 98% for HPV types 16 and 18 and 68.4% for the 10 most frequent non-vaccine HPV types (HPV-31/33/35/39/45/51/52/56/58/59) related to CC, based on the clinical trial results of the AS04-adjuvanted HPV-16/18 vaccine, with weights reflecting the relative frequency of the different HPV types in Nigerian women , an Objective function:Subject to the following constraints:xi ∈ ℝWith xi is a continuous decision variable representing the proportion of the population in strategy i; these four values for xi are the optimization variables i  that denote the four different predefined prevention strategies: no prevention, screening once, vaccination alone, and vaccination plus screening once.■ s denotes a subset of the i strategies including screening .■ v denotes a subset of the i strategies including vaccination .■ xnprev represents, among the xi, the proportion of the population receiving no preventive measure.■ Coverage1 and Coverage2 denote the upper bounds for coverage for screening and vaccination, respectively; these are selected to represent either readily achievable or ideal values.■ CCi represents the number of CC cases at steady state per 100,000 women receiving prevention strategy i as estimated by the evaluation model.■ bi is the cost for 100,000 women at steady state receiving strategy i as estimated by the evaluation model.■ B is the overall CC-related (prevention and treatment) budget across the population.■ The pre-vaccination budget was estimated assuming that there is no national screening or vaccination program in Nigeria, the associated expenditure per woman per year being the cost of treatment for those with CC, corresponding to $0.25 per woman per year across the entire female population. In the base-case analyses, the constraint on annual expenditure per woman was increased gradually from $0.25 to $2.0, and the annual incident number of CC cases was estimated for each level of annual expenditure when the optimal combination of prevention strategies is implemented. For the two vaccination schedules 3 and 2 doses were considered. Three scenarios were estimated for the full range of budget constraints: (1) maximum screening coverage of 20% and maximum vaccination coverage of 95%; (2) maximum screening coverage of 40% and maximum vaccination coverage of 95%; and (3) maximum screening coverage of 20% and maximum vaccination coverage of 50%. These ranges were selected based on expected or targeted ranges for screening and vaccination coverage within the Nigerian setting. An additional constraint required a minimum number of people to receive no prevention. This constraint equals the lower of one minus the upper-bound coverage constraint for either screening or vaccination and hence is directly linked to the screening and vaccination coverage constraint.Univariate sensitivity analyses were conducted to estimate the effects on the incident number of CC cases for two different budget constraints, set at $1 and $2 per woman per year, when changing the costs associated with screening, vaccination, and treatment of CIN and CC. Ranges of plus or minus 20% were used for the screening costs, and ranges of plus or minus one standard deviation from the means were used for the costs of treatment of CIN and CC, using the data from the retrospective chart review. In addition, the impact on CC cases of adding strategies with more frequent screening, two lifetime screenings (at ages 35 and 40 years) or three lifetime screenings , was tested. The sensitivity analysis on treatment costs accounted for setting with higher or lower costs than the one estimated in the retrospective cost evaluation that may not be representative of all settings in Nigeria. An additional scenario included the possibility of implementing an HPV test screening instead of the Pap test. In this scenario the cost of the screening test was set to 50% of the Pap test costs and CIN sensitivity was increased by an absolute value of 10%. Finally, the duration of vaccine efficacy was reduced from lifetime to 25 years, and also the vaccine efficacy was reduced by an absolute value of 20%.Table Figure With a budget constraint of $2.00 per woman per year, the maximum prevention coverage could be reached and the optimal mix of prevention strategies to minimize CC incidence would be (with both a 2- and a 3-dose vaccination schedule), 75% with vaccination alone, 20% with vaccination and one lifetime screening, and 5% with no prevention strategy. This would result in a CC reduction of 64% with both a 3-dose and a 2-dose vaccination schedule. The resulting expenditure would be $1.93 (3-dose vaccination schedule) and $1.35 (2-dose vaccination schedule) per woman per year.Although the most effective of the four strategies included in the base case is vaccination plus one lifetime screening, the optimal mix of prevention strategies does not include this combination until the budget constraint per woman is set to $2.00 with a 3-dose vaccination schedule and $1.50 with a 2-dose vaccination schedule.Figures With a 3-dose vaccination schedule Figures ,4 and6One way sensitivity analyses were performed for a budget constraint of $1 and $2 per woman per year. The costs of screening and treating CIN grade 1, CIN grades 2 and 3, and CC were varied, as was the frequency of lifetime screenings and the duration and level of protection resulting from vaccination. An additional scenario investigated the use of a HPV test as the screening method instead of the Pap test. The results of the sensitivity analyses, measured as the percentage of CC cases prevented compared with the pre-vaccination incidence of CC cases  are shown in Table With a $2 per woman budget constraint, the maximum vaccination and screening coverage is reached under the optimal mix of strategies for all sensitivity analyses performed, and hence the maximum CC reduction is reached with both the 3-dose and the 2-dose vaccination schedule scenarios and under all sensitivity analyses.We have developed a model that would identify the optimal mix of CC prevention strategies (screening and or vaccination) to minimize the number of CC cases for different scenarios defined by constraints on budget, maximum screening and vaccination coverage, and overall reachable population. Under the base case, three scenarios were considered for either a 3-dose or a 2-dose potential vaccination schedule to capture multiple alternatives regarding the available budget and feasibility of implementation in Nigeria.The results of the Markov evaluation models indicated that the number of CC cases expected from a 100% coverage of each prevention strategy was lowest for vaccination  compared with one lifetime screening , two lifetime screenings , three lifetime screenings , or no prevention .In the base-case optimization model analyses, with upper-bound coverage constraints of 20% for screening and of 95% for vaccination and a budget constraint at $1 per woman, the optimal mix of prevention strategies would result in a 31% CC reduction compared with today’s CC incidence with a 3-dose vaccination schedule, and in a 46% CC reduction with a 2-dose vaccination schedule. With a 3-dose vaccination schedule, the optimal combination would be 20% with screening alone, 39% with vaccination alone and 41% without any prevention, while with a 2-dose vaccination schedule the optimal combination would be 0% screened, 71% vaccinated, and 29% without any prevention. Under the lower budget constraints, the optimal strategy with a 3-dose vaccination schedule would always be a combination of screening alone, vaccination alone and no prevention, while with a 2-dose vaccination schedule the optimal combination would only include vaccination and no prevention by screening. Using an increment in budget constraint of $0.25 per women per year going from $0.25 to $2.00, any budget constraint equal to or higher than $2.00 per woman with a 3-dose vaccination schedule, or $1.50 per woman with a 2-dose vaccination schedule, would result in the optimal strategy with a maximum CC reduction using a strategy consisting of 75% with vaccination alone, 20% with vaccination and screening and 5% without prevention. The associated CC reduction would be 64%. These strategies would be specifically associated with a budget of $1.93 per woman with a 3-dose vaccination schedule and $1.35 per woman with a 2-dose vaccination schedule.The results were similar for different screening and vaccination coverages, except that in the case of an upper-bound vaccination coverage of 50%, the percentage reduction in CC cases compared with the pre-vaccination situation had a maximum value of 35% at an expenditure of $1.18 per woman with a 3-dose vaccination schedule and $0.88 per woman with a 2-dose vaccination schedule. Sensitivity analyses indicated that the results were sensitive to the CIN and CC treatment cost. Higher treatment costs resulted in lower achievable coverage with vaccination and screening within the $1 budget constraint, and hence a lower reduction in the number of CC cases. With a 2-dose vaccination schedule, all sensitivity analyses tended to first maximize the vaccination coverage. With a 3-dose vaccination schedule, most sensitivity analyses (except a high CIN treatment cost) tended to maximize both vaccination and screening and in all cases resulted in a lower reduction in CC than the reduction obtained with a 2-dose vaccination schedule.The results from the optimization model indicate that when introducing a prevention program for CC, in a country like Nigeria where none is currently in place, with a 2-dose vaccination schedule it would be optimal to invest in vaccination alone to the extent feasible, followed by vaccination plus one lifetime screening. With a 3-dose vaccination schedule, a combination of both vaccination and screening would be optimal. This result depends therefore on the relative costs of vaccination and screening as well as the relative efficacy between the two interventions. The optimization would favor a strategy with a low costs and a high efficacy (sensitivity for the screening).A similar model was recently used to estimate the optimal mix of CC screening and HPV vaccination in the UK and Brazil. These tThe current evaluation estimated that an optimal allocation of a CC prevention budget of $1 per woman per year could lead to an important reduction in CC burden by 31% to 46%, depending on the scenario chosen. In Nigeria, three tiers of Government  have responsibility for health and decide about budget allocation. The current evaluation suggests that a combination of 2-dose vaccination with or without one lifetime screening is expected to lead to an important reduction in CC.Nationwide deployment of HPV vaccines to reduce the CC burden in Africa is more realistic than implementing a screening program, given the current failure of the national screening program due to logistic and funding challenges. Most African countries have efficient and very well accepted national childhood immunization programs, as in Nigeria. HPV vaccination could be integrated into these programs, provided some operational challenges linked to the adaptation of the program to a different target population (adolescent for the HPV vaccination) are addressed. The cost of implementation would also be drastically reduced compared to that expected from the implementation of a screening program. However, in each country the age at which the vaccination is implemented should be adapted based on their national average age of girls’ sexual debut.The optimization model used for this analysis has its limitations. First, the model outcome is obtained when an optimal mix of different prevention strategies is applied in the population and a steady-state incidence of CC is achieved across the population. Many years will elapse before reaching that state. The results therefore represent an optimal “ideal” situation that provides a direction to be followed but not a result that will occur immediately, and nor does it address specific implementation issues. Vaccination will affect the girls who will become the women of the future. Today’s women would not benefit from vaccination, but could directly benefit from a single lifetime screening. The vaccinated girls would not need any screening in the years post vaccination. This cost saving could be used to implement a screening program benefiting older women. This would accelerate the overall CC reduction. No discounting was applied in this analysis, as the model presents a steady-state evaluation over a 1-year period. Given the time lag needed to reach steady state, discounting could be considered, but the means of implementation and especially the value of including a discount rate in such an optimization exercise is debatable. A discounting applied to the input of the model down to a today value would not impact the optimization results while ways to implement the optimization on a population overtime still need to be investigated.Second, a static Markov model was used to evaluate the cost and outcomes of each potential strategy at steady state across a population over a 1-year period. As the optimization model takes results from the evaluation model, the robustness of the optimization results therefore also depends upon the validity of the evaluation model. The Markov model used for this analysis did not account for dynamic effects of vaccination on infection, such as herd protection. It also assumed stability in the population demographic structure over time. However, using a dynamic model as the evaluation model instead of the static model would be more difficult to adapt to countries with limited data. It would also lead to nonlinearity between coverage and the outcomes, and thus would require the use of a nonlinear programming optimization model.Third, the models presented here are deterministic: costs and effects in all combinations are known. It would be possible to characterize the allocation problem under conditions of uncertainty by reformulating the model as a stochastic analysis. However, existing approaches for such stochastic analyses are limited. This isFourth, the results depend on the strategies initially investigated using the evaluation model. Therefore, including a vaccine with a different profile or a screening program with different characteristics  may lead to other optimal scenarios, as presented in the sensitivity analysis conducted in the current evaluation. Furthermore, the analysis does not take into account the possibility that the introduction of vaccination may affect the sensitivity of the screening test,51. It aThe optimization model presented here provides information needed to inform health policy decisions on the optimal allocation of limited resources to prevent CC most effectively. The results indicate that increased spending will be needed in Nigeria and in other countries in Sub-Saharan Africa that currently lack a CC prevention program, in order to achieve adequate prevention of CC cases. With a 3-dose vaccination schedule, the most efficient allocation of a limited budget would be to invest in both vaccination and screening, while with a 2-dose vaccination schedule investment should first be directed to vaccination before implementing a screening program.All costs are in USD ($ in the text).CC: Cervical cancer; CIN: Cervical intraepithelial neoplasia; CIN1: Cervical intraepithelial neoplasia, grade 1; CIN2/3: Cervical intraepithelial neoplasia, grades 2 and 3; HPV: Human papillomavirus; NGN: Nigerian naira; NS: No solution found; PAHO: Pan American Health Organization; Pap: Papanicolaou; PV: Pre-vaccination schedule; SD: Standard deviation; TNM: Classification of Malignant Tumours; UK: United Kingdom; VE: Vaccine efficacy; WHO: World Health Organization; 2LT: Two lifetime screenings; 3LT: Three lifetime screenings.IFA received financial support for conference attendance and speaking engagements from the GlaxoSmithKline group of companies and is serving on an Independent Data Monitoring Committee for an International HPV study.ND is an employee of the GlaxoSmithKline group of companies and owns restricted shares in the GlaxoSmithKline group of companies.IOMB and BA declare no conflict of interest.ND conceived and designed the study, developed the model, conducted the analyses and was involved in the review and discussion of the results. IOMB, BA and IFA collected the data and were involved in the scheme analysis, review and discussion of the results. All authors reviewed and commented on drafts, and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/365/prepubOptimal mix of strategies under the optimal budget allocation. (a) vaccination overage 95%, screening coverage 20%; (b) vaccination coverage 95% screening coverage 40%; (c) vaccination coverage 50% screening coverage 20%. Sensitivity Analyses: Budget Constraint $1 (~4 times Pre-vaccination Budget) per Woman. CC = Cervical cancer; 2LT = Two lifetime screenings; 3LT = Three lifetime screenings; HPV = Human papillomavirus; VE = Vaccine efficacy.Click here for fileOptimal mix of strategies under the optimal budget allocation. (a) vaccination overage 95%, screening coverage 20%; (b) vaccination coverage 95% screening coverage 40%; (c) vaccination coverage 50% screening coverage 20%. Sensitivity Analyses: Budget Constraint $2 (~8 times Pre-vaccination Budget) per Woman. CC = Cervical cancer; 2LT = Two lifetime screenings; 3LT = Three lifetime screenings; HPV = Human papillomavirus; VE = Vaccine efficacy.Click here for file


We performed a randomised trial in very preterm, small for gestational age (SGA) babies to determine if prophylaxis with granulocyte macrophage colony stimulating factor (GM-CSF) improves outcomes . GM-CSF was associated with improved neonatal neutrophil counts, but no change in other neonatal or 2-year outcomes. As subtle benefits in outcome may not be ascertainable until school age we performed an outcome study at 5 years.280 babies born at 31 weeks of gestation or less and SGA were entered into the trial. Outcomes were assessed at 5 years to determine neurodevelopmental and general health status and educational attainment.We found no significant differences in cognitive, general health or educational outcomes between 83 of 106 (78%) surviving children in the GM-CSF arm compared with 81 of 110 (74%) in the control arm. Mean mental processing composite  at 5 years were 94 (SD 16) compared with 95 (SD 15), respectively (difference in means −1 (95%CI −6 to 4), and similar proportions were in receipt of special educational needs support ). Performance on Kaufmann-ABC subscales and components of NEPSY were similar. The suggestion of worse respiratory outcomes in the GM-CSF group at 2 years was replicated at 5 years.The administration of GM-CSF to very preterm SGA babies is not associated with improved or more adverse neurodevelopmental, general health or educational outcomes at 5 years.ISRCTN42553489. Neonatal neutropenia and sepsis are common following fetal growth restriction.Administration of granulocyte macrophage colony stimulating factor (GM-CSF) after birth raises neutrophil counts but does not improve neonatal or 2-year developmental outcomes.Granulocyte macrophage colony stimulating factor (GM-CSF) administered after birth does not alter 5-year neurocognitive outcomes.Preterm small-for-gestational-age (SGA) babies have a higher prevalence of special educational needs than anticipated from their gestational age at birth.Perinatal infection and inflammation have been implicated in the causal pathway of neonatal preterm brain injury and deviations from normal neurodevelopmental patterns.Very preterm babies with fetal growth restriction, who are born small for gestational age (SGA), are at high risk of developing acquired sepsis after birth and this has been believed to relate to the frequent observation of neutropenia in such babies. Granulocyte macrophage colony stimulating factor (GM-CSF) has been shown to be effective at reducing neutropenia-related infections in patients with cancer after chemotherapy.As part of the original design of the trial, we hypothesised that there might be more subtle benefits over and above the relatively short-term developmental outcomes described above and therefore designed an outcome evaluation at 5 years of age to determine whether the administration of GM-CSF in the neonatal period was associated with differences in the prevalence of developmental problems.Full details of the PROGRAMS trialhttp://www.sdqinfo.com) to evaluate behaviour at school.Contact was maintained with the families enrolled in the study following the 2-year evaluation; parents were informed of the results of the trial and follow-up study by newsletter. As previously, the children were traced and the families contacted by the study coordinator. Parents were asked for permission to carry out the evaluation at 5 years and to provide details of their child's school. Head teachers were then approached and a request made for a member of the study team to visit the school and carry out the testing. Parents were invited to attend as they wished. The assessment was carried out as close to the child's 5th birthday as feasible and no adjustment was made for gestational age at birth. Where a child was in a special school with serious disability, a formal assessment by a paediatrician and estimate of learning attainment was made. Details of school attainment and need for extra educational support  were provided by the child's class teacher, who also completed the teacher's report for the Strengths and Difficulties Questionnaire  of the Kaufmann-ABC (Pearson UK).9In addition to the SDQ, parents also completed questionnaires detailing household socioeconomic status, and the language and communication element of the 4-year evaluation from the Twins and Early Development Study,11The sample size for the initial study was based on the short-term primary outcome, survival without sepsis for 14 days from trial entry.4A CONSORT diagram was constructed, showing the flow of participants through the study.13All analyses were conducted on an intention-to-treat basis, that is, participants were never excluded from analyses on the basis of the treatment received. The participants lost to follow-up or withdrawn were not included in analyses.Written informed consent was obtained from parents. Trial oversight was provided by an independent steering committee and independent data monitoring and ethics committee. This study is registered as an International Standard Randomised Controlled Trial, number ISRCTN42553489.Of 280 babies enrolled in the study, 279 completed the study intervention, 64 babies died , 11 were withdrawn by their parents and a further 41 families were lost to follow-up at 5 years. Details of the participant flow through the study are shown in In those who were followed up, the two study groups were balanced over a range of social and demographic measures (see web tables S2 and S3). The index of multiple deprivation scorePrimary outcome: Kaufmann MPC scores for the GM-CSF group were 94 (SD 16) compared with 95 (SD 15) in controls, difference in means: −1 (95%CI −6 to 4). Subscale scores did not vary significantly between groups and the proportion scoring −2 SD or lower (<70) or between −1 and −2SD were similar . There wFewer families returned health questionnaires than the number of children who were assessed at school: 62 in the GM-CSF group and 54 in the control group (see web table S4). Admissions to hospital were infrequent in both groups and no child was admitted to an intensive care unit and mechanically ventilated. At 5 years the point prevalence of cough and wheeze were higher and the use of respiratory medications more frequent in the GM-CSF group , but none of the comparisons would be statistically significant if adjusted for multiple comparisons.This is the first report of childhood outcomes at 5 years of age following the neonatal administration of GM-CSF to SGA very preterm babies in a randomised trial. We have previously reported that GM-CSF raises neonatal neutrophil counts, but does not appear to reduce the prevalence of neonatal sepsis or other neonatal morbidities; nor affect neurodevelopment at 2 years of age adjusted for prematurity.For the 5-year outcomes we achieved reasonable follow-up rates of 76%. Surviving children who were not evaluated did not differ over a range of perinatal and neonatal variables from children evaluated as part of this study. Nonetheless children who are not available for follow-up may have higher rates of impairment compared with those evaluated,We chose a wide range of neuropsychological tests from direct observation, parents and teachers to make a comprehensive assessment. Furthermore we obtained summary functional information concerning educational attainment to confirm the results of our testing. Although the general cognitive scores appear only mildly depressed compared with standardisation data (5–6 points or approximately 0.33 SD), the test used was standardised in the late 1970s.In the absence of a comparison group of babies born at full term it is difficult to evaluate the performance of this group of SGA children relative to other very preterm groups. The majority of children in this study had fetal growth restriction. In a prospective cohort study of children identified before birth with growth restriction, scores at 6 years were on average 11 points lower compared with AGA-matched children, whose IQ scores were 12 points lower than term-born children.The persistence of the trend for increased risk of respiratory symptoms is intriguing. Cohort studies associate bronchopulmonary dysplasia with perinatal infectionPrior to the clinical use of G-CSF and GM-CSF in newborn babies during the 1990s, concern was raised that exposure of the immature haematopoietic and immune system to pharmacological doses of these recombinant human cytokines might lead to later alternations to haematopoiesis, immune function, or drive the emergence of malignant clones and leukaemia.27When evaluated in a randomised trial, with long-term follow-up at 2 years and 5 years of age, GM-CSF does not appear to offer significant benefit in terms of neuroprotection.


Drosophila. A well-established energy sensor is the insulin signaling (InS) pathway. Previous studies in various animal models including human have revealed the role of insulin levels in short-term memory but its role in long-term memory remains less clear. We therefore investigated genetically the spatial and temporal role of InS using the olfactory learning and long-term memory model in Drosophila. We found that InS is involved in both learning and memory. InS in the mushroom body is required for learning and long-term memory whereas long-term memory specifically is impaired after InS signaling disruption in the ellipsoid body, where it regulates the level of p70s6k, a downstream target of InS and a marker of protein synthesis. Finally, we show also that InS is acutely required for long-term memory formation in adult flies.Memory formation has been shown recently to be dependent on energy status in  Drosophila  in tissue growth is well established  or rational (slow) thinking  and are labeled by their accession number. Wild-type, OK107GAL4, ELAVGAL4, FEB170GAL4, and 232GAL4 were obtained from Dr. Tim Tully (Cold Spring Harbor Laboratory). UAS-Chico was obtained from Dr. Minoru Saitoe. Insulin DN is from Bloomington Stock center (8252). All stocks were homogenized with the WT background [w1118 (isoCJ1)] by backcrossing for 6 generations.In all cases virgin females of the GAL4 driver or wild type flies were collected and crossed to the UAS-transgene males.Drosophila were raised after being dechorionated at 22-23°C. Adult flies 1-3 days old were used for the behavior experiments. Flies were trained as described previously for learning and memory  and 4-methylcyclohexanol (MCH) with relative concentration adjusted for equal averseness. A footshock is paired for 60 s with either other and then the paired odor is reversed in the subsequent trial. The shock consists of twelve 1.25 s pulses of 60 V DC with 5 s interpulse interval. The flies are provided with fresh air between the 2 odors for 45 s. After giving the second odor, the flies are again given 45 of fresh air before being moved to the elevator. 90 s later the flies are brought to the choice point where they are exposed on 1 side with the odor that was paired with the shock and on the other with the odor not paired with the shock. Flies are given 2 min to do their choice before being trapped and counted. For learning, a single training session is used. For spaced training, the flies receive the same training session without the testing part but it is repeated 10 times with 15 min rest interval between each training session. Flies are allowed to rest for 24 h and tested after that. For massed training, flies are given 10 training sessions also but this time without rest interval. For each performance index (PI), there is a component of shocking to each odor which allows to balance any slight odor preference. The PI is calculated as the average fraction of the number of flies avoiding the shocked odor minus the fraction of flies avoiding the non-shocked odor. So, if flies distribute 50:50, the PI would be zero.T-test was used. Statistics were performed with Prism and JMP. Comparison with one asterisk are significant to P < 0.05, two asterisks indicate P < 0.001 and three asterisks P < 0.0001. All graphs depict ± s.e.m.For experiments where multiple groups are present, a One-Way ANOVA followed by a Tukey post test was performed. For experiments where 2 groups are compared, Student To make sure that the observed defects were not related to lack of olfaction or shock sensation or impairment with locomotion, the flies were tested for those modalities as well. Olfactory acuity and shock reactivity were assessed as in Boynton and Tully  and DuraAcute heat-shock was performed as in Yin et al.  and moreOne to three days old female flies were dissected and processed as described previously. Bolduc et al.  Flies arFor FAS-II staining, we used 1d4 antibody (1:200) from Developmental Hybridoma study bank. We used the midline crossing phenotype established by Michel et al as reference. For p70S6K Thr398 we used Cell signaling (9209S) at a concentration of 1:25. All images within a given panel were acquired with the same gain to allow for comparison. Quantification was performed using ImaJ. The brain region was selected for the measurements. We used Cy3 anti-mouse as secondary antibody (Jackson Immuno Laboratory) at concentration of 1:200.For the experiments with training, flies were trained as usual to MCH. In the untrained group, flies were presented with the odors as usual but without receiving the shock paired to the odors. Imaging was performed at 1 day after spaced training or mock spaced training.chico mutant flies, we sought to see if long-term memory was also affected by InS. We started by focusing on chico, the only Drosophila homolog of the insulin receptor substrate (IRS). We first expressed RNAi against the Drosophila insulin receptor substrate, chico, pan-neuronally using ElavGAL4. We were able to replicate the previous findings from Naganos et al.  was significantly defective. Moreover, we found that long-term memory  was also significantly affected  and 1-day memory after spaced training     Next, we tested the performance at 1 day after spaced training of the InR RNAi or DN expressing flies and observed a significant defect in long-term memory for both   . In addition, we noted that the basal level of protein synthesis in the ellipsoid body was markedly decreased compared to control flies  Figure . Similar) Figure . Similar) Figure . The sam) Figure . There w) Figure . In addi) Figure . We founs Figure . Overexps Figure  In summachico mutants learning defect by expressing chico in the mushroom bodies. We therefore started by testing the effect of insulin receptor (InR) and chico disruption in the mushroom bodies   also led to significant learning defects. Memory after spaced training was also impaired with disruption of either chico or InR  but had no effect on memory after massed training   Figure . Similar Figures  or senso Figures .DN in the central complex  or InR   is sufficient to trigger transient expression of HSP70GAL4  nor chico  led to defects in immediate learning  or the UAS-chico  when compared to flies without expression  and the insulin receptor substrate (chico) are required for normal learning and protein synthesis dependent memory but not for protein synthesis independent memory. We show that InS in the ellipsoid body, a region highly connected and compared to the basal ganglia (Lin et al., The brain has the highest energy requirement of all organs, reaching 87% of the resting metabolic rate in childhood (Kuzawa et al., DC and FB conceived and designed the project and analyzed the experiments. DC, FB, SL, and CR performed the behavior experiments. DC, AA, and FB wrote the paper with comments from the other authors. AA and MH performed the immunohistochemistry experiments.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.


Although the application of patient-specific instruments (PSI) for total knee arthroplasty (TKA) increases the cost of the surgical procedure, PSI may reduce operative time and improve implant alignment, which could reduce the number of revision surgeries. We report our experience with TKA using PSI techniques in 120 patients from March to December 2014. PSI for TKA were created from data provided by computed tomography (CT) scans or magnetic resonance imaging (MRI); which imaging technology is more reliable for the PSI technique remains unclear. In the first 20 patients, the accuracy of bone resection and PSI stability were compared between CT and MRI scans with presurgical results as a reference; MRI produced better results. In the second and third groups, each with 50 patients, the results of bone resection and stability were compared in MRI scans with respect to the quality of scanning due to motion artifacts and experienced know-how in PSI design, respectively. The optimized femoral cutting guide design for PSI showed the closest outcomes in bone resection and PSI stability with presurgical data. It is expected that this design could be a reasonable guideline in PSI. Total knee arthroplasty (TKA) has high success rates with the majority of patients experiencing rapid improvement in pain, function, and quality of life . AccuratThese theoretical advantages have been postulated but have not been confirmed in the literature to date . It is aTherefore, the purpose of this study is (1) to evaluate the effect of PSI design in CT and MRI with respect to secure fit and bone cutting, (2) to compare bone cutting in different PSI designs with respect to motion artifacts in MRI, (3) to compare bone cutting with respect to optimized PSI designs, and (4) to develop a preoperative plan and resultant custom guides that could accurately replicate surgeon preference with infrequent intraoperative changes.2 .This retrospective cohort study was approved by the institutional review board of our hospital. All patients provided informed consent before surgery. Between March and December 2014, we included 120 patients with end-stage knee osteoarthritis scheduled for TKA in the study. Patients with rheumatoid arthritis, previous osteotomy, fractures, retained hardware in the limb, or claustrophobia were excluded. The inclusion criteria were diagnosis of primary knee osteoarthritis and the ability to undergo MRI at our facility. Mean patient age was 70.9 years  and mean body mass index (BMI) was 27.4 kg/mFor the 20 patients in the first group, both CT and MRI (1 mm and 2 mm slice thickness) images were taken for accuracy comparisons. CT images of the knee joint were taken with a slice interval of 1 mm using a 64-channel CT scanner , whereas 5 mm slice thickness was applied to hip and ankle joints. The tube parameters were 120 kVp and 135 mA. The acquisition matrix was 512 × 512. The field of view was 200 mm.MRI images were acquired using a 1.5T MRI scanner . MRI scans of the tibiofemoral knee joint were obtained at 1 or 2 mm slice thickness in the sagittal plane, whereas 5 mm slice thickness was applied to hip and ankle joints in the axial plane. For the nonfat saturation condition, MRI consisted of an axial proton-density (PD) sequence. A high-resolution setting was used for the spectral presaturation inversion recovery sequence . All procedures were identical to those in Signature from Biomet.The first author (Oh-Ryong Kwon) had participated in 30 TKA operative cases with Signature at the time of the study. Signature charges $900 to fit each patient's unique anatomy and to guide surgical bone resection in order to manufacture femoral and tibial PSI guides . The tim3D data can be acquired through either MRI or CT. The 3D reconstruction processes were performed with Mimics software . Using Mimics, the resulting 3D images were converted to STL files and implemented in the digital CAD software, 3-Matic, also produced by Materialise. 3-Matic allows the user to combine geometry from mixed sources into a single project. PSI guides were designed with 3-Matic commercial software .Patients from the first group were divided into two different groups with respect to slice thickness, 1 mm from CT, 1 mm from MRI, and 2 mm from MRI scans . CT has limitations in delineating articular cartilage (3D model inaccuracies) , 12. Carμm accuracy. The distal femur resections were measured medially and laterally, thus obtaining distal femur medial and lateral resection measurements. Posterior femoral condyle cuts were made and resections in the medial and lateral posterior femoral condyles were measured. Bone cutting data was comparatively analyzed with preplanning results. The thickness of the saw blade was added to the resection thickness to calculate the total resection for each cut.Each step was evaluated for each group and intraoperative changes were recorded, including resection level, component size, and coronal/sagittal alignment. The resected bone was then measured with a 3D laser scanner  with 50 All operations followed by TKA surgical preplanning were conducted by an experienced surgeon (Oh-Ryong Kwon). A computer-generated preoperative plan was created according to the surgeon's preferences, as follows: default alignment for femoral component rotation was parallel to the surgical epicondylar axis, femoral component coronal alignment 90 degrees to the mechanical axis, and femoral component sagittal alignment 3 degrees of flexion with 9.5 mm distal medial resection. We retained the default plan when it appeared appropriate and recorded all changes when made. The time from submission of the 3D image to receipt of the guides was 4 days. All patients in both cohorts received a posterior-stabilized, fixed-bearing implant. The operation was performed through an anteromedial parapatellar approach, without everting the patella. Cement fixation was used in all patients. The implant used was the Genesis II Total Knee System .The results presented are the intraoperative changes for measurements of femoral component size and alignment in groups 1, 2, and 3. The differences between preplanning results and actual bone cutting were measured in groups 2 and 3.One hundred and twenty patients underwent an operation with custom-fit technology. n = 12) of femurs. PSI predicted the varus and valgus alignment and internal and external rotations in 96% (n = 5) and 89% (n = 13), respectively, of femurs.Intraoperative changes to the implant sizing and alignment proposed by PSI were observed (groups 1–3). A total of 94 intraoperative changes were made in 120 TKAs (0.8 changes per knee) with the use of PSI . In group 1, for the 13 patients within the subgroup with 1 mm slice thickness MRI, the time spent in the MRI machine was long and impatience led to motion artifacts. Therefore, bone models and PSI guides could not be developed and manufactured due to the inaccuracy of the scans. In group 1, the PSI cutting guides that were manufactured using 1 mm slice thickness MRI scans were used for only one of 20 patients. Similarly, in group 1, the PSI cutting guides based on CT scans and 2 mm slice thickness MRI scans were used for four and fifteen patients, respectively.Throughout the path from group 1 to group 3, the frequency of intraoperative changes of the implant size and alignment decreased. In group 3, the percentage of changes was 3% (The mean (±standard deviation) discrepancies between the predicted and actual resection thicknesses (“cutting error”) are shown in The most important finding of this study was the optimal medical imaging method and critical thickness for MRI slices for the design of PSI and the development of the optimal design for a PSI guide well-fitted to the patient's anatomy without any micromotion in contact with the bony surface.The outcome of TKA greatly depends on the surgical technique used , 14. TecWe hypothesized, based on the experience using Signature PSI systems, that inaccuracy in 3D models and PSI guides not perfectly fitted to bone are the main two reasons for the resulting dissimilarities between preplanned outputs and measured bone resections in the operating room.It is prohibited for the hospital to charge patients for PSI in Republic of Korea. Preplanning and design processes were conducted using previously validated commercial software from Materialise , 21, andIn this study, the difference between CT and MRI scans was analyzed first. There is a lack of soft tissue modeling in the models based on CT scans; thus the contact surface was considered to be bone . Even foThe required scan time for MRI with 1 mm slice thickness is about 70% more than that of MRI with 2 mm slice thickness. There are many motion artifacts disturbing the design of PSI guides in the 1 mm slice MRI scan, especially in elderly patients . For thiFor CT based PSI guides, it did not provide a good stability due to the absence of soft tissue such as articular cartilage and contact using spike for the gap between guide and bone. MRI based PSI guides designed without regard to patient's motion artifact could lead to its instability in contact with bony surface. For MRI image with motion artifact, it was found from group 1 that it might lead to the more inaccurate result if full contact PSI design is applied. If there is uncertainty in cartilage regions due to motion artifact in MRI, it was found that tolerance in design may produce better stability in contact of PSI guide with bone. Therefore, 2 mm slice thickness conditions were used in the group 2 and group 3 designs.For motion artifacts in group 2, the areas with cartilage were considered contact surfaces, and, if not, tolerance was applied in the design. If there were no motion artifacts, the PSI design was shape-matched following the articular surface. We developed the optimal design for PSI through the learning curve related to cases with motion artifacts. PSI design with only anterior and distal contact regions in group 2 constrained flexion and extension. The current Signature PSI guides which were similarly designed from other companies could not tightly hold internal-external rotation. Therefore, we considered the optimal design to overcome this problem by holding flexion-extension and internal-external rotation fixed. Throughout such modifications, not only translation but also rotational stability was improved during guide drilling. Throughout the process from group 1 to group 3, the intraoperative changes in alignment and implant sizing diminished.Bone resection was measured with a 3D scanner, instead of a 2D micrometer, to obtain more precise qualitative measurements. The resection cutting results reflect how well PSI-TKA surgery was completed after preoperative planning. To our knowledge, this is the first research paper to evaluate the design of PSI guides with respect to their contact stability and relation to 3D medical images. The overall discrepancy was small for the resection thicknesses compared to the preplanned results (groups 2-3). Thus, we are satisfied with the PSI that were planned. However, it is worth noting that greater variation was noted in the distal femoral resections, which were found to be decreased in the optimal design. The significant finding in this study was that group 3 with motion artifacts and full contact design and group 2 without motion artifacts had similar discrepancies between the predicted and actual resection. In other words, design improvement may compensate for the negative effect of motion artifacts on MRI quality.A complete patient-specific system can potentially shorten operative time, setup time, operating room space, and hospital space. Setup time is shortened by eliminating the need to bring and open multiple instrumentation sets in the operating room. Procedural time can also be reduced in the complete patient-specific system that includes all instrumentation and a patient-specific implant by eliminating several time-consuming steps. When the instrumentation and implants are completely patient-specific, the implant sizing, rotation, and positional decisions are predetermined. These implant attributes either can be based on a standard set of design rules or could be customized according to surgeon preference.There were limitations to our study. First, this current study did not assess final implant positioning, which will be investigated in future research. Second, a single experienced surgeon made all the preplanning and intraoperative decisions concerning changes to alignment and implant sizing. This is not representative of the decisions of a low-volume or inexperienced knee surgeon using this technology. A future study with multiple surgeons would provide a more comprehensive representation of this technology based on surgeon experience. One surgeon working on the planning by himself could reduce confusion, compared to working with multiple surgeons . FinallyIn conclusion, the optimum PSI design for stability improvement was suggested. In terms of 3D image scanning time required, this would be beneficial to the hospital and also to the patients. The approach outlined introduces a generic product in addition to the commercial PSI systems offered by other manufacturers. In the future, various PSI design methods should be evaluated for variable conditions ranging from different MRI systems to patient anatomy.


Reliable mouse models are key in the discovery and development of novel anticancer treatments. Non-invasive monitoring techniques such as bioluminescence imaging (BLI) are useful tools to determine tumor engraftment and evaluate tumor growth. However, the development of ascites in ovarian cancer mouse models leads to possible difficulties. Ascites can interfere with the set-up of correct end points and can interfere with the evaluation of tumor volume using BLI. We provide optimized euthanasia criteria and in vivo data underlining the pitfalls of BLI. With great interest, we read the article of Liao et al. entitled “Preservation of tumor-host immune interactions with luciferase-tagged imaging in a murine model of ovarian cancer”  in the JEstimating and monitoring tumor load in ovarian cancer is challenging. With innumerable peritoneal implants that are formed, we rely in a clinical setting on CT (computed tomography) or MRI (magnetic resonance imaging) to evaluate tumor growth and disease progression, based on the RECIST 1.1 criteria . The REC6 ID8-fLuc cells), long before macroscopic tumor or weight increase is detected. For further reference we will refer to the experiments performed by our research group with Firefly luciferase (luc1) transduced ID8 cell line as ID8-fLuc in contrast to ID8-Luc2 described by Liao et al. However, according to us, the technique also comes with significant shortcomings: is the photon flux a reliable measure for tumor volume after the onset of ascites  to the mice [6 ID8-fLuc cells ip the mice developed clinically appreciable ascites 6 weeks after inoculation, in contrast to the group inoculated with 10 × 106 ID8-fLuc cells, which developed ascites 8 weeks after tumor engraftment  at day 80. This suggests that there is no improved immune control of tumor growth in the transfected cell line, on the contrary, if anything the ID8-fLuc cell line grows faster in vivo when compared to the parental cell line.The authors spent a lot of effort in studying the changes in the humoral and cellular immunity due to the Luciferase insert. This is highly valuable information, especially for the use of the model in immunotherapy research. It is reassuring to see that although serum Luciferase IgG increases, it does not seem to have a detrimental influence on tumor growth represented by the BLI-signal. After transfection these cells express not only firefly Luciferase, but also the resistance protein  on which they are selected. This protein is also a possible source of immunogenicity of the tumor. However, we would like to point out that in our model we noticed an increase in the in vivo tumor growth after stable lentiviral transduction and selection compared to the parental wild type ID8 (ID8-WT) model. As depicted in Fig. In the article of Liao et al. FACS analysis was performed on ascites, spleen and tumor of ID8-WT inoculated mice compared to ID8-Luc2 inoculated mice at 12 to 15 weeks after inoculation to evaluate the tumor microenvironment. These are late stage mice in which immunosuppression has completely taken over from immune control. These results prove that in late stage animals there is no difference in the microenvironment due to the expression of luc2, but do not allow us to draw conclusions concerning early disease, when there is still an equilibrium between immune control and immune escape. Furthermore to determine the change in in vivo tumor growth due to the Luciferase insert, it would be more relevant to flank the firefly Luciferase cDNA with loxP sites, allowing Cre mediated excision of the cassette once the stable cell line is established, providing a perfect control. When comparing the cell line with and without the addition of Cre recombinase, the only difference is the presence firefly Luciferase and not the selection pressure that has been put on the cells during the transfection and selection process.In conclusion, the use of codon-optimized firefly luciferase Luc2 expressed in ID8 cells as described by Liao et al. is suitable for the evaluation of tumor load in early stage disease. Care should be taken in the interpretation of BLI results once ascites occurs. When using overall survival as an outcome measure in this model we recommend repeated ascites drainages to avoid underestimating survival. The use of orthotopic, immune competent models for ovarian cancer should be encouraged, as they are an adequate representation of the clinical setting of ovarian cancer patients.