Microcystis aeruginosa during growth with low levels of dissolved inorganic nitrogen (low N), low levels of dissolved inorganic phosphorus (low P), and in the presence of high levels of high molecular weight dissolved organic matter (HMWDOM). Under low N, one third of the genome was differentially expressed, with significant increases in transcripts observed among genes within the nir operon, urea transport genes (urtBCDE), and amino acid transporters while significant decreases in transcripts were observed in genes related to photosynthesis. There was also a significant decrease in the transcription of the microcystin synthetase gene set under low N and a significant decrease in microcystin content per Microcystis cell demonstrating that N supply influences cellular toxicity. Under low P, 27% of the genome was differentially expressed. The Pho regulon was induced leading to large increases in transcript levels of the alkaline phosphatase phoX, the Pst transport system (pstABC), and the sphX gene, and transcripts of multiple sulfate transporter were also significantly more abundant. While the transcriptional response to growth on HMWDOM was smaller , transcripts of multiple genes specifically associated with the transport and degradation of organic compounds were significantly more abundant within HMWDOM treatments and thus may be recruited by Microcystis to utilize these substrates. Collectively, these findings provide a comprehensive understanding of the nutritional physiology of this toxic, bloom-forming cyanobacterium and the role of N in controlling microcystin synthesis.Whole transcriptome shotgun sequencing (RNA-seq) was used to assess the transcriptomic response of the toxic cyanobacterium  Microcystis which produces the hepatotoxin microcystin Microcystis have been associated with higher temperatures Microcystis bloom populations have been correlated with inorganic P concentrations MicrocystisMicrocystisMicrocystisMicrocystisMicrocystis cultures One of the most common bloom-forming cyanobacteria in temperate freshwater ecosystems is Microcystis blooms are associated with high levels of nutrient loading Microcystis blooms may rely on organic N and P compounds for nutrition. In aquatic ecosystems, dissolved organic nitrogen (DON) can exceed 50% of the total N pool Microcystis is able to grow using urea or amino acids such as alanine, leucine, and arginine as N sources Microcystis to utilize refractory forms of DON, however, is poorly understood. With regards to organic forms of P, previous research has demonstrated that cyanobacteria can assimilate and utilize organic compounds such as phosphomonoesters and phosphonates While Microcystis aeruginosa has yielded important insight regarding the genetic potential of this cyanobacterium Aureococcus anophagefferens while Straub et al. M. aeruginosa genes displayed significant changes in their transcript abundance during the transition between the light and dark cycle. While investigations of how Microcystis responds to differing nutrient concentrations and sources at the transcript level have yet to be performed, the availability of a fully annotated genome makes such an approach tractable. The goal of this study was to obtain a comprehensive understanding of the nutritional physiology of Microcystis. Cultures of Microcystis aeruginosa (clone LE-3) were grown with low N, low P, high molecular weight dissolved organic matter (HMWDOM), and under ideal conditions while nutrients levels, cellular physiology, and gene expression patterns were assessed using whole transcriptome shotgun sequencing (RNA-seq).The sequencing of the complete genome of Microcystis aeruginosa clone LE-3  et alMicrocystis bacteria in cultures. Cultures were maintained in modified BG-11 medium  illuminated by a bank of fluorescent lights that provided a light intensity of ∼100 µmol quanta m−2 s−1 on a 14∶10 light/dark cycle at 21°C.Experiments were performed with M. aeruginosa LE-3 grown under low N and P, and on HMWDOM. Experimental treatments included a control in which triplicate cultures were grown with replete amounts of N and P ) and five treatments including low N (75 µM nitrate), low P (0 µM Pi), HMWDOM  as the sole N and P source, a treatment with HMWDOM supplemented with 1.26×10−3 M Pi (HMWDOM+P), and a treatment with HMWDOM supplemented with 8.0×10−5 M ammonium (HMWDOM+N). The low N and low P treatments were used to explore transcriptional responses to N and P stress and thereby identify genes responding solely to low N (increased transcripts in the low N treatment only), genes responding to solely low P (increased transcripts in the low P treatment only), and those that responded as a consequence of a reduced growth (increased transcripts in both the low N and low P). The HMWDOM treatment was used to explore transcriptional responses when cultures were grown exclusively on organic substrates. The HMWDOM treatments supplemented with either N or P were used to assess what genes might be transcribed by M. aeruginosa to acquire organic forms of P or N, respectively, from HMWDOM.Transcriptomic profiling experiments were performed to assess the full transcriptional response of Microcystis blooms HMWDOM was isolated via tangential flow filtration (TFF) 5 cells mL−1 and monitored for cell densities, in vivo chlorophyll a fluorescence, photosynthetic efficiency, alkaline phosphatase activity (APA), and dissolved nutrient concentrations (see below for methods) at the same time daily to avoid diel changes in gene expression and cell physiology i, nitrate, and ammonium <0.6, 3.6, and 0.8 µM, respectively), suggesting cultures were utilizing organic compounds for growth. At the time of harvest, 50 mL aliquots of each replicate in each treatment were centrifuged for 10 min. at 2,500×g at 21°C. The supernatant was poured off and resulting cell pellet was resuspended with 1 mL of remaining media and placed into a 2 mL microcentrifuge tube. The concentrated sample was centrifuged again for 10 min. at 2,500×g at 21°C and immediately flash frozen in liquid nitrogen and stored at −80°C. The entire harvest process took <30 minutes per experimental flask.Triplicate cultures were inoculated with 3×10free™ kit according to the manufacturer’s instructions. The concentration and quality of total RNA was assessed with an Agilent Bioanalyzer™. Ribosomal RNA was removed from total RNA (∼2µg) using a Ribo-Zero™ rRNA Removal Kit for Gram-Negative Bacteria (Epicentre®) according to the manufacturer’s instructions and samples were then again reassessed with a Bioanalyzer to verify rRNA removal. Enrichment of mRNA was conducted using a TruSeq™ RNA Sample Preparation Kit v2 (Illumina®) according to the manufacturer’s instructions. Sequencing was performed by the JP Sulzberger Columbia Genome Center  with an Illumina HiSeq 2000 System and ∼ 8 million, 100bp, single end reads per replicate sample were mapped to a reference genome Microcystis aeruginosa NIES-843 p≤0.05) of these changes to be assessed http://blast.ncbi.nlm.nih.gov) was used to elucidate putative functions of hypothetical genes. The Illumina sequences reported in this paper have been deposited in the National Center for Biotechnology Information’s Sequence Read Archive (accession no. SRP021202).Total nucleic acids were extracted using the CTAB technique 2/N1)/(t2–t1) where N1 and N2 equal the biomass at time 1 (t1) and time 2 (t2) respectively et alphoX) in Microcystis aeruginosa LE-3 (p<0.005) in vivo (Fi) and DCMU -enhanced in vivo fluorescence (Fm) of each replicate experimental sample on a Turner Designs TD-700 fluorometer (EM filter of >665 nm and EX filter of 340–500 nm). All readings were blank corrected using BG-11 media. DCMU blocks electron transfer between PSII and PSI and yields maximal fluorescence and previous studies have demonstrated that Fv/Fm can be a sensitive diagnostic of nutrient limitation, reaching a maximal value of ∼ 0.65 under nutrient replete conditions, and decreasing to less than half of that under nutrient limitation Lugol’s iodine preserved cells were enumerated using a Beckman Coulter Multisizer™ 3 Coulter Counter® with a 50 µm aperture which allowed cell densities to be quantified with a relative standard deviation of 3%. Cell densities of selected samples were verified microscopically with a hemacytometer. Growth rates were calculated for each day of the experiment based upon changes in cell abundance according to the equation µ = Ln and regressions were performed using SigmaPlot version 11.0 (build 11.1.0.102). Post hoc multiple comparisons were performed with Tukey Tests and data sets which did not meet the assumption of normality or heterogeneity of variance were log transformed prior to analyses.p<0.05, Tukey Test; p<0.05, Tukey Test; −1 hr−1) compared to the control  whereas those grown on low N and low P were significantly lower , depending on the treatment  with 1,080 genes having significant increases in transcript abundance relative to the control and 930 genes having significant decreases in transcript abundance relative to the control  were involved in photosynthesis and respiration (20%) as well as hypothetical functions  and nitrite reductase (narB) were 8.9-fold and 7.8-fold more abundant under low N conditions (urtABCDE) had significant increases in transcripts with the exception of the urtA gene (glnA) displayed a 6.6-fold increase in transcript abundance. In addition, 19 ABC transporters displayed significant increases in transcript abundance under the low N conditions whereas many of the P acquisition and transport genes displayed significant decreases in transcript abundance  had fold change values ranging from 1.4 to 10  also decreased in transcript abundance with fold change values ranging from 3.4- to 10-fold (nblA) displayed a dramatic 50-fold increase in transcript abundance (rbcL and rbcS respectively) and genes involved in ATP synthase, carbon dioxide concentrating, and NAD(P)H dehydrogenase subunits  decreased transcript levels by 15-fold  had large, 17–49-fold increases in transcript abundance (i transport system (pstABC) increased 1.8- to 49-fold (phaE gene (MAE 50040), which is involved in the production of polyhydroxybutyrate (PHB), an energy storage molecule produced by microbes in response to physiological stress, also displayed a significant increase in transcript abundance under P deficiency , representing 27% of the s genome . Many ofs genome . Four gebundance . In addi 49-fold . Other g 49-fold . FurthercpcA2 and cpcB2) as well as two additional cytochrome c oxidase subunit genes (ctaDI and ctaEI). In contrast, two genes encoding for large and small subunit ribulose biphosphate carboxylase (rbcL and rbcS) had similar decreases in transcript levels to those observed in the low N treatment  largely fell within the “hypothetical” (on average 32%) and “unknown” (on average 17%) functional categories  increased in abundance. When cultures were grown on HMWDOM+inorganic N, the alkaline phosphatase gene, phoX, increased transcript levels 3.1-fold as did three inorganic phosphate binding and transport genes  increased, on average, 2.6-fold  displayed large increases in transcript abundance when grown on HMWDOM relative to the control, with the highest fold change values observed in the cultures grown on HMWDOM and HMWDOM+P  increased in the HMWDOM and HMWDOM+P treatments by 25- and 17-fold, respectively , and a protease gene (MAE 19290) increased by 1.5-fold in the HMWDOM treatment  had lower transcript levels in all treatments with the lowest level observed in the low N treatment  accumulates intracellularly, stimulating the transcription of ntcA which in turn induces transcription of N assimilation genes Anabaena and Synechococcus, the nir operon that encodes nitrate and nitrite reductases and transporters (nirA-nrtABCD-narB) is expressed at high levels when ammonium is not present in the growth medium Microcystis cultures which were deprived of inorganic N and this trend was most evident in cultures grown on HMWDOM where transcript levels of these genes were nearly triple that of when grown with low N, a finding consistent with the low levels of inorganic N present in these cultures. In some cyanobacteria, the presence of ammonium in the growth medium represses the expression of the nir operon Microcystis and/or that the presence of HMWDOM had a stronger effect on transcription of this operon.Under low N, Microcystis transcriptional patterns in low N cultures differed from expectations. For instance, while the annotation for Microcystis aeruginosa NIES-843 indicates that it has two ferredoxin-nitrite reductase genes , only one was expressed when cultures were deprived of N  further downstream of the expressed nirA gene that also increased in transcript abundance under low N conditions. Assuming these genes are acting upon nitrate/nitrite, this further illustrates the complex arrangements of nitrate/nitrate assimilation genes within Microcystis, a finding consistent with studies of other cyanobacteria Some urtBCDE), suggesting these genes are transcribed in response to low N conditions. Curiously, many of the urease encoding genes (ureABCDEFG) had decreased or unchanged transcript levels when under low N or in the presence of HMWDOM, except for two urease accessory proteins E and F which displayed significant increases in transcripts in low N, low P, HMWDOM, and HMWDOM+P. These findings are consistent with those observed in Anabaena in which the expression of the urtABCDE genes increased during N-limitation but the genes encoding urease did not Microcystis as has been reported for some microbes Urea is produced by the decomposition of organic compounds, is a waste product excreted by many animals, and can be found at micromolar concentrations in the natural environment Microcystis cultures displayed elevated rates of alkaline phosphatase activity and transcripts for genes within the Pho regulon, including pstABC, pstS, sphX, phoX, and phoU which were all present at high levels, a finding consistent with other cyanobacteria MicrocystispstS and pstABC genes had increases in transcript levels although these increases varied between 1.8 and 49-fold. The largest fold change was observed in the pstS gene, MAE 18310 . Microcystis, like Synechocystis PCC 6803 pst gene clusters (MAE 18340 to 18380 and MAE 09250 to 09280) but also has an additional pstS gene (MAE 18310) and three additional pstA genes (MAE 18280 to 18300) positioned downstream of one of the pst gene clusters M. aeruginosa LE-3 was grown with low P, the pst gene cluster represented by MAE 18340 to 18380 had large transcript increases (4.6–21-fold change) while the second gene cluster and additional pst genes displayed smaller increases (1.8–3.6-fold change). This illustrates the dynamic and varied response of Microcystis in the face of P-stress. We originally reported that M. aeruginosa NIES-843 did not have the alkaline phosphatase gene phoASynechococcus elongatus PCC6301 phoA . This gene, however, did not increase transcript levels under low P as did the phoX gene suggesting that, if it is an alkaline phosphatase, it is less active.Under P-stress, sqd gene set and in cyanobacteria, the sqdX gene is the most likely candidate to encode sulfolipid synthase Microcystis was grown with low P, 5 of 7 sulfate transporters displayed significant increases in transcript abundance suggesting heightened requirement for sulfur within the cell. Transcripts of these transporters were significantly lower in all other treatments. There was, however, no change in sqdX gene transcript abundance under low P, while these transcripts decreased in abundance when cells were starved for N (Low N and HMWDOM+P treatments). Collectively, these findings may indicate that the P-deprived cells were actively sequestering sulfur but had not begun replacing their phospholipid membranes with sulfolipids Microcystis have yet to be identified.Under low P conditions, some cyanobacteria substitute sulfur in place of phosphorus within their membrane lipids Synechococcus sp. strain PCC 7942, the degradation of phycobiliproteins was found to be partial under P-stress or complete under sulfur- or N-stress and the gene facilitating phycobiliprotein degradation has been identified as nblAM. aeruginosa LE-3 displayed significantly lower photosynthetic efficiency and large transcriptional increases of the nblA gene when grown on low levels of inorganic N (Low N and HMWDOM+P) and significant but smaller transcriptional increases during P-limitation (low P and HMWDOM+N). Furthermore, nutrient stress generally reduces photosynthetic rates in aquatic primary producers Microcystis.When cyanobacteria are starved for an essential nutrient, they undergo chlorosis as photosynthetic pigments and phycobiliproteins are degraded and photosynthetic rates decline Aureococcus anophagefferens can grow on HMWDOM as a sole N source, depleting >25% of the available N from these compounds and displaying high rates of cell surface peptide hydrolysis Alexandrium tamarense, was able to use N from riverine HMWDOM as efficiently as nitrate Microcystis on HMWDOM substrates.In the absence of inorganic forms of N and P, aquatic primary producers may rely on organic compounds for these elements. A variety of studies have explored the use of urea and amino acids by cyanobacteria M. aeruginosa LE-3 was grown on HMWDOM, there were low levels of dissolved inorganic nitrogen (DIN) and dissolved inorganic phosphorus (DIP), elevated APA, and depressed photosynthetic efficiency relative to the control suggesting the cultures were physiologically stressed by the low levels of N and P present in cultures. There was a smaller transcriptional response to HMWDOM by Microcystis suggesting it has fewer unique biochemical pathways for directly utilizing organic compounds compared to the wider array of pathways recruited to grow under low N and P. For instance, among genes uniquely expressed in a single treatment, there were 491 and 333 such gene transcripts present at significantly different abundances under low N or low P, respectively, whereas the numbers of unique transcripts present at significantly different levels when grown on HMWDOM, HMWDOM+N, and HMWDOM+P were smaller  and the ammonium permease gene, amt1, suggesting that Microcystis responded to HWMDOM in a manner similar to N-stress, even when there was excess ammonium present (HMWDOM+N). When grown on HWMDOM, Microcystis had more genes in common with low N and HMWDOM+P than any other treatment suggesting a stronger response to N-stress than P-stress (phoX as well as the Pst Pi transport system (pstABC), and the sphX, phosphate transport gene, demonstrating that the addition of N stimulated P-stress in cultures grown with HWMDOM.The transcriptional responses of P-stress . With reSynechocystis sp. PCC 6803  and an ABC transporter in the C39 peptidase family (MAE 54550) with putative roles in cleaving double-glycine leader peptides from bacteriocins Microcystis  and has been annotated as having similarity to an ATPase in Trichodesmium erythraeum (strain IMS101). Gene MAE 51260 is most similar to a putative lipopolysaccharide transport protein in other strains of Microcystis  which may be involved in transporting carbohydrates, organic alcohols, and acids Some genes with higher transcript abundances in various treatments were likely important for transporting and metabolizing N or P from organic compounds. For instance, the gene MAE 55030, which encodes for a protein within the CHAT peptidase domain and is important for protein degradation Growth rates are linked with a number of biochemical processes in phytoplankton and can be influenced by temperature, nutrient availability, and light Microcystis spp colonies Microcystis, this cyanobacteria was exclusively responsible for the transcript data presented in this study.Heterotrophic bacteria are known to inhabit the mucilage of Microcystis cell was significantly reduced and transcripts of the microcystin synthetase genes (mcy cassette) were less abundant suggesting that microcystin synthesis is dependent upon a sufficient N supply. Some of these genes also had decreased transcript levels in the HMWDOM and HMWDOM+P treatments that also had low levels of inorganic N, affirming the importance of high N levels for microcystin synthesis , and the sphX gene. These global analyses revealed that Microcystis has two pst gene clusters (pstSCAB), additional copies of the pstA and pstS genes, and a phoA gene that was not expressed, suggesting a broad and active, yet complex, P harvesting ability. Additionally, genes encoding for sulfate binding and permease proteins had increased transcript levels under low P, while genes involved in photosynthesis had lower transcript levels. In contrast to N and P, there was a smaller transcriptional response to growth on HMWDOM. In many cases, the transcriptional patterns of the HMWDOM with N or P treatments tended to mirror the low P and low N treatments, respectively. There were, however, transcripts of multiple genes specifically associated with the transport and degradation of organic compounds that were significantly more abundant within HMWDOM treatments that may be important for utilizing organic compounds.In summary, whole transcriptome sequencing of Microcystis aeruginosa LE-3 grown with low N, low P, and HMWDOM is a first step toward providing a broader understanding of the nutritional physiology of this toxic, bloom-forming cyanobacterium. Future studies of this species may target a finer scale time series of its transcriptomic response or additional strains of this species. Future proteomic studies will help verify transcriptional patterns observed in this study and future ecosystem studies will be useful for assessing the extent to which the transcriptional patterns displayed by cultures are manifested within wild populations of Microcystis during bloom events.The transcriptomic profile of Figure S1Daily changes in Microcystis cell densities during experiments. Red boxes indicate the day cells were harvested for transcriptomic sequencing.(TIFF)Click here for additional data file.Figure S2Fold change in transcripts of genes involved in nitrogen metabolism. Bars represent the fold change in gene expression relative to the control treatment (p≤0.05).(TIFF)Click here for additional data file.Figure S3Fold change in transcripts of genes involved in phosphorus metabolism. Bars represent the fold change in gene expression relative to the control treatment (p≤0.05).(TIFF)Click here for additional data file.Figure S4Fold change in transcripts of genes involved in sulfate binding and transport within the low P treatment. Bars represent the fold change in gene expression relative to the control treatment (p≤0.05).(TIFF)Click here for additional data file.Table S1Summary of transcriptomic sequencing results.(XLSX)Click here for additional data file.Table S2Statistical differences in gene expression. Values represent the number of genes and percentages represent the percent of the total differentially expressed genes.(XLSX)Click here for additional data file.Table S3Differential expression results for the low N treatment compared to the Control treatment.(XLSX)Click here for additional data file.Table S4Genes within the photosynthesis and respiration functional category. Columns C through G contain fold change values relative to the control with positive and negative values as described in (XLSX)Click here for additional data file.Table S5Differential expression results for the low P treatment compared to the Control treatment.(XLSX)Click here for additional data file.Table S6Differential expression results for the HMWDOM treatment compared to the Control treatment.(XLSX)Click here for additional data file.Table S7Differential expression results for the HMWDOM+N treatment compared to the Control treatment.(XLSX)Click here for additional data file.Table S8Differential expression results for the HMWDOM+P treatment compared to the Control treatment.(XLSX)Click here for additional data file.Table S9Genes showing differential expression in all treatments. Columns B through F contain fold change values relative to the control with positive and negative values as described in p≤0.05.(XLSX)Click here for additional data file.Table S10Genes showing similar differential expression in the low N and low P treatments. Columns B through F contain fold change values relative to the control with positive and negative values as described in (XLSX)Click here for additional data file.


S. Typhimurium SL1344, and its dependence on the bacterial alarmone, guanosine tetraphosphate (ppGpp) during late stationary phase, (LSP). Under LSP conditions we were able to identify the transcriptional start sites (TSSs) for 53% of the S. Typhimurium open reading frames (ORFs) and discovered 282 candidate non-coding RNAs (ncRNAs). The mapping of LSP TSSs enabled a detailed comparison with a previous dRNA-seq study of the early stationary phase (ESP) transcriptional architecture of S. Typhimurium SL1344 and its dependence on ppGpp. For the purposes of this study, LSP was defined as an aerobic LB culture grown to a later optical density reading (OD600 = 3.6) compared to ESP (OD600 = 2.3). The precise nucleotide positions of the majority of S. Typhimurium TSSs at LSP agreed closely with those identified at ESP. However, the identification of TSSs at different positions, or where additional or fewer TSSs were found at LSP compared to ESP enabled the genome-wide categorisation of growth phase dependent changes in promoter structure, the first time such an analysis has been done on this scale. Comparison of the ppGpp-dependency LSP and ESP TSSs for mRNAs and ncRNAs revealed a similar breadth of ppGpp-activation and repression. However, we note several ncRNAs previously shown to be involved in virulence were highly ppGpp-dependent at LSP. Finally, although SPI1 was expressed at ESP, we found SPI1 was not as highly expressed at LSP, instead we observed elevated expression of SPI2 encoded genes. We therefore also report an analysis of SPI2 transcriptional architecture at LSP resulting in localisation of SsrB binding sites and identification of a previously unreported SPI2 TSS. We also show that ppGpp is required for nearly all of SPI2 expression at LSP as well as for expression of SPI1 at ESP.We have used differential RNA-seq (dRNA-seq) to characterise the transcriptomic architecture of  Salmonella are one of the most frequent causes of food-borne illness worldwide. Although it is difficult to estimate precisely due to unreported incidents, a recent report suggests that there are 93.8 million cases of gastroenteritis due to Salmonella species globally each year with 155,000 deaths Infections caused by non-typhoidal S. Typhimurium, which mainly causes a self-limiting enterocolitis but can cause a more serious disease in immunosuppressed patients S. Typhimurium is able to penetrate intestinal epithelium cells, a process that is dependent on the expression of a type 3 secretion system (T3SS) encoded by a horizontally acquired set of virulence genes encoded within Salmonella Pathogenicity Island 1 (SPI1). In the case of immunocompromised humans, S. Typhimurium can become systemic resulting in a typhoid-like fever due to its ability to replicate and survive within macrophages; this is facilitated by the intracellular expression of a second T3SS encoded within SPI2 S. Typhimurium during stationary phase. Stationary phase occurs when nutrients become scarce and bacteria develop a multiple-stress resistant state. Morphological changes are observed, including rounded shape, loss of flagella and thickening of the cell wall. General metabolism is redirected, macromolecular degradation is increased, and storage and osmoprotection compounds are synthesized In the present study we focus on Mycobacterium tuberculosisListeria monocytogenesLegionella pneumophiliaVibrio choleraPseudomonas aeruginosaS. Typhimurium, virulence in Balb/C mice was shown to be completely dependent on ppGpp due to the presence of SpoT rather than RelA S. Typhimurium at ESP during expression SPI1 and its dependence on ppGpp, has previously been defined using dRNA-seq resulting in the definition of TSSs for the majority of S. Typhimurium genes and the discovery of many new ncRNAs S. Typhimurium and its dependence on ppGpp during later stationary phase when the expression of SPI2 encoded genes are elevated, in addition to changes in the expression patterns of many other genes and ncRNAs involved in the transition to a nutrient deprived and microaerophilic environment.Guanosine tetraphosphate is produced by the RelA and SpoT enzymes in all beta- and gammaproteobacteria and whereas RelA only has ppGpp synthetic function, SpoT is able to both synthesise and hydrolyse ppGpp (for reviews see S. Typhimurium during early stationary phase (ESP) identified the TSSs for 78% of the S. Typhimurium genome S. Typhimurium genome during LSP and show that the majority of LSP TSS positions agree to within 0–3 nt of our previously defined ESP TSSs. However, we found that 13.5% of the LSP TSSs were present at LSP and not ESP or had differently positioned TSSs compared to previously defined ESP TSSs S. Typhimurium at stationary phase was found to be highly ppGpp-dependent with 33% of the TSSs for protein coding genes and 29.4% of the candidate LSP ncRNAs being directly or indirectly controlled by ppGpp.Our previous analysis of the transcriptomic architecture of S. Typhimurium parental strain, SL1344, and an isogenic ΔrelAΔspoT strain grown aerobically to LSP in LB. The respective growth curves for the parental and ΔrelAΔspoT strains were shown to be almost identical (600 = 3.6) and time at which samples were taken for dRNA-seq analysis are indicated in http://jbrowse.org/) by following the link provided at www.ifr.ac.uk/Safety/MolMicro/.A dRNA-seq approach was used to identify the precise nucleotide positions of TSSs from RNA samples isolated from an dentical . The grorelAΔspoT strain; however many had multiple associations and these are defined and summarised in We mapped a total of 2186 LSP TSS's on to the SL1344 chromosome , although sequencing reads extended into both ORFs implying that under the growth conditions used here, they were co-transcribed with ssrA , as might be expected to occur in a stationary phase microaerophilic environment. At LSP, lack of nutrients is also a limiting growth factor and this was suggested by the presence of LSP TSSs for genes encoding gluconate, glucose and iron transporters . A low phosphate and magnesium environment is suggested by the presence of LSP specific TSSs for pstB, which encodes a phosphate transport ATP-binding protein rstB which is the sensor kinase in a two component system (with RstA) rstB is induced under low magnesium growth conditions through the PhoPQ two-component system S. Typhimurium and has been shown to be involved in modulating Salmonella biofilm formation pstB and rstB has also been shown to trigger activation of SPI2 under minimal media growth conditions The LSP mRNA TSSs were compared to our previously identified ESP TSSs , S2. AltA comparison of the TSSs at LSP and ESP found within the same promoters was performed and revealed that 134 TSSs either differed in position (>10 nt) and/or were found to have fewer or more TSSs at LSP compared to ESP . For eashilC and hilD and also the SPI1 encoded genes prgH, and invH , slyA and virK. Similarly we could also identify 70 genes at LSP where the ESP TSS was absent but where an LSP TSS at a differing position was present , pagC, ygdP and rfaH. Lrp has been shown to affect many stationary phase induced genes including those involved in the response to nutrient limitation, high concentrations of organic acids, and osmotic stress S. Typhimurium ygdP have been shown to reduce invasion of Salmonella by repressing SPI1 expression E. coli pathogens and has also been shown to down-regulate key virulence factors in S. Typhimurium Interestingly several genes in which one or more ESP TSSs were absent at LSP (and therefore presumably not used at LSP) had virulence related functions; these included the SPI1 regulators ssaB, ssaC, ssaV and sseG), and orf32. Other virulence related genes with candidate asRNAs included virK, stcD, mgtB and two genes related to iron transport: iroC and fhuE (E. coli σ70 (−10) binding site (TATAAT), , . An analTATAAT), . We weregltA which encodes citrate synthase (relAΔspoT strain and ppGpp-repressed expression was defined as 4-fold or higher transcript levels in the ΔrelAΔspoT strain compared to the parent strain (relAΔspoT datasets. Only 96 (5.2%) TSSs were found to be ppGpp-activated by 4-fold or greater. In total 33% of the TSSs for protein coding genes were ppGpp-dependent at LSP compared to 34% at ESP; together these are indicative of the major role ppGpp plays in modulating the stationary phase gene expression programmes   .For the stable RNA genes, the TSSs for all but 2 of the operons containing rRNA genes were ppGpp-repressed (greater than 4-fold), consistent with the ppGpp-repression of TSSs for genes involved in translation/protein modification described above, and with the general down-regulation of protein synthesis occurring at LSP . HoweverIt is known that ppGpp-repressed promoters tend to contain GC rich discriminator regions between the TSS and −10 regions that play a role in destabilising the RNAP- promoter complex resulting in transcriptional repression  were ppGpp-repressed by a factor of 4-fold or higher; this is more than double the total proportion of ppGpp-repressed ESP ncRNAs 10.2%, . We alsoS. Typhimurium at later stationary phase resulted in the prediction of TSSs for 53% of ORFs which were found to closely correlate with the TSS positions for these ORFs previously defined at ESP The definition of the transcriptomic architecture for ssaR. We were unable to identify a TSS upstream of ssrB or orf242, suggesting they were co-transcribed with ssrA  which were opposite the 5′ UTRs of ahpC and entC repectively, the former encodes a subunit of alkyl hydroperoxide reductase involved in combating oxidative stress and the latter encodes the bacterial siderophore enterochelin, involved in iron uptake within the host S. Typhimurium virulence  were located in the intergenic regions of genes and we note that 16 and 3 candidate ncRNAs were opposite the 5′ and 3′ regions of genes respectively and hence may play a irulence . SdsR sRvice versa. These observations support evidence suggesting that although ppGpp plays a major role in transcriptional regulation, its action is essentially passive and modified by other regulatory elements such as sigma factors hilA or rtsA and neither could we identify a TSS for hilD in the parental strain at LSP . We note that this TSS was also identified as ppGpp-repressed at ESP with very similar read numbers compared to LSP relAΔspoT strain compared to the parent strain , conditions previously shown to induce SPI2 gene expression relAΔspoT strain, and time of harvesting are shown in The virulent S. Typhimurium parent strain, SL1344 and the isogenic Δhttp://www.vertis-biotech.com) as described in relAΔspoT non-enriched (DM-NE) and ΔrelAΔspoT enriched (DM-EN). Each library was tagged with a different barcode at the 5′ end to enable multiplexing during sequencing. Four of the Illumina-Solexa libraries were pooled and sequenced in a single lane using 50 single-read cycles on an Illumina Genome Analyzer II sequencing machine , yielding a total of ∼2×107 reads.In order to differentiate primary from processed transcripts, total RNA from biological replicates of each strain were divided into equal quantities and one half treated with Terminator 5′-phosphate-dependent exonuclease  as previously described S. Typhimurium SL1344 genome (Genbank ID: FQ312003.1) including the three native virulence plasmids  using the segemehl program The mapping statistics for the Illumina-Solexa cDNA libraries are shown in Figure S1S. Typhimurium SL1344 parental and ΔrelAΔspoT strains.Growth curves for (DOCX)Click here for additional data file.Figure S2Single promoter rearrangements at LSP compared to ESP. Red arrows indicate LSP-specific TSSs.(DOCX)Click here for additional data file.Figure S3MEME analysis of 282 candidate LSP ncRNAs showing conserved -10 region (TATAAT).(DOCX)Click here for additional data file.Figure S4gltA transcriptional start sites.ppGpp-dependent location of (DOCX)Click here for additional data file.Figure S5Promoter architecture and ppGpp-dependency of SPI1.(DOCX)Click here for additional data file.Figure S6S. Typhimurium SL1344 parent and ΔrelAΔspoT strains from microarray transcriptomic data.Heat map showing clustered expression levels of genes encoded within SPI1 and SPI2 from early to late stationary phase in (DOCX)Click here for additional data file.Table S1S. Typhimurium SL1344 ORFs at LSP.Master table of TSSs and ppGpp-dependent expression levels for (XLSX)Click here for additional data file.Table S2TSSs with absent or altered positions at LSP compared to ESP.(XLSX)Click here for additional data file.Table S3TSSs and ppGpp-dependent expression of candidate LSP ncRNAs.(XLSX)Click here for additional data file.Table S4Expression levels and ppGpp-dependent expression rRNAs and tRNAs at LSP and ESP.(XLSX)Click here for additional data file.Table S5relAΔspoT libraries.Mapping statistics for SL1344 parental and Δ(XLSX)Click here for additional data file.Table S6Location of SsrB binding sites relative to transcription start sites at SsrB dependent promoters.(DOCX)Click here for additional data file.Table S7ESP TSSs with no corresponding LSP TSS or alternative TSS.(XLSX)Click here for additional data file.Table S8TSSs and ppGpp-dependent expression of known and predicted sRNAs that have been validated by Northern blot analysis.(XLSX)Click here for additional data file.


It has been shown that vitamin D deficiency is associated with autoimmune diseases, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), multiple sclerosis (MS) and type 1 diabetes (T1DM), and that vitamin D supplementation prevents the onset and/or development of these autoimmune diseases . However3 levels by the competitive protein binding assay . Vitamin D deficiency in our study was defined as levels of 25(OH)D3 below 15 ng/ml, as previously described [P < 0.05 was considered statistically significant.A total of 26 female patients with newly onset GD were consecutively recruited from the subjects who attended the Sumire clinic to check the thyroid disease in winter and spring periods in 2011. GD was diagnosed by clinical and biochemical symptoms of hyperthyroidism, the presence of diffused goiter and TRAb positivity. Forty-six control healthy female subjects who had normal thyroid function without antithyroid peroxidase antibody (TPOAb) and antithyroglobulin antibody (TgAb) were recruited in the same period. This study was approved by the ethical committee of Sumire clinic, and informed consent was obtained from all patients. Vitamin D status was evaluated by the measurement of serum 25(OH)DClinical characteristics in GD patients and control subjects were shown in Table 3 levels were significantly lower in GD patients compared to control subjects . The prevalence of vitamin D deficiency in GD patients was significantly higher compared to control subjects . Serum 25(OH)D3 levels were significantly associated with serum calcium levels  and intact parathyroid hormone levels  in GD patients. In addition, there was significant association between serum 25(OH)D3 levels and thyroid volume , but not thyroid function and TRAb levels in GD patients. Although further study would be necessary to conclude, these results suggest that the vitamin D status may be involved in the pathogenesis of GD.To examine an involvement of vitamin D in the pathogenesis of GD, we evaluated the vitamin D status in female patients with newly onset GD and the association of serum vitamin D levels with various GD-related clinical factors. As shown in Table To our best knowledge, this is the first report showing the decreased vitamin D levels in newly onset GD patients and the significant association between vitamin D status and thyroid volume, a clinical factor related to the pathogenesis of GD. Because it has been reported that vitamin D status is not changed by the treatment for GD , it is u+ T cell response, vitamin D directly inhibits the production of Th1 cytokines (IL2 and IFN-γ), and enhances Th2 cytokine (IL-4) production [Vitamin D is known for its primary role in bone and mineral homeostasis, and it has been shown recently that its deficiency is associated with various diseases such as cardiovascular disease, cancer, infection, and adiposity as well as osteoporosis . Interesoduction –7. FurthGD is an autoimmune thyroid disease in which TSH receptor autoantibodies cause hyperthyroidism. The pathogenesis of GD is complicated and involved in multiple genetic and environmental factors. It has been thought that GD occurs with the infiltration of T cells in the thyroid gland as a result of these factors. Subsequently, T cells elaborate various cytokines leading to the production of TSH receptor autoantibodies. Especially, helper T cells produce various cytokines, including IFN-γ which induces thyrocytes to express major histocompatibility complex class II surface HLA-DR antigens and renders them susceptible to immunologic attack. Although HLA-DR antigens are not normally expressed on thyroid cells, in GD, the thyroid follicular cells present HLA-DR antigens on their surface with potential involvement in triggering autoimmune process . Recent In conclusion, vitamin D levels in female patients with newly onset GD are decreased and significantly associated with thyroid volume. It is noted, however, this study is cross-sectional survey with a small number of subjects, and limited in its ability to conclude that vitamin D status is directly related to the pathogenesis of GD. Therefore, the direct role of vitamin D in patients with GD should be examined by further prospective clinical studies by the treatment of vitamin D and experimental studies.


Transcription activator-like effector nucleases (TALENs) are widely used in recent years in gene targeting for their specific binding to target sequences when engineered in vitro. Here, we show that the repair of a double-strand break (DSB) induced by TALENs adjacent to a TR can produce serial types of mutations in the TR region. Sequencing analysis revealed that there are three types of mutations induced by the DSB repair, including indels only within the TR region or within the flanking TALEN target region or simutaneously within both regions. Therefore, desired TR mutant types can be conveniently obtained by using engineered TALENs. These results demonstrate that TALENs can serve as a convenient tool for modifying TRs in the genome in studying the functions of TRs.Tandem repeats (TRs) are abundant and widely distributed in eukaryotic genomes. TRs are thought to have various functions in gene transcription, DNA methylation, nucleosome position and chromatin organization. Variation of repeat units in the genome is observed in association with a number of diseases, such as Fragile X Syndrome, Huntington's disease and Friedreich's ataxia. However, the underlying mechanisms involved are poorly understood, largely owing to the technical limitations in modification of TRs at definite sites in the genome  Even years before the nucleotide sequencing methods were developed, a great mass of repeated sequences were detected in vertebrate genomes by denaturation-renaturation experiments in vivo.TRs can distribute everywhere in the genome, including gene bodies, untranslated regions (UTRs) and promoters Xanthomonas in recent years in vitro by fusing TAL effector with Fok I nuclease, so that TALENs can recognize specific DNA sequences (by TAL effector) followed by creating a double strand break (DBS) in the target site (by Fok I nuclease). In living cells, DSBs can be repaired in two pathways Transcription activator-like (TAL) effectors are sequence-specific DNA binding domain proteins identified from plant pathogenic bacteria Danio rerio) used in our experiments is long-fin strain. Bisexual diploid goldfish (C. auratus) and unisexual polyploid goldfish (C. auratus pengze) were purchased from nearby farms and maintained in our laboratory in the breeding season. Artificial spawning and fertilization were performed as previously reported Zebrafish (no tail (ntl), a decisive developmental regulatory gene, was obtained from the nucleotide database in EMBL. The upstream sequence of goldfish ntl was obtained after four rounds of genome walker using GenomeWalker™ Universal Kit . The gene-specific primers (GSPs) used are listed in Table S1  Fok I nuclease functions as a dimer when used to make double-strand breaks and the length of the spacer can affect the specificity of TALENs, TALENs were designed in pairs that bind opposing DNA target sites separated by a spacer, and the length of the spacer can affect the specificity of the TALRNs The range of spacer length used for searching potential targets was between 12-18bp, within the suggested region of optimal activity for TALENs A (TG)Spe I and Nhe I  and then cloned into pCS2-Fok I plasmids .The engineered TAL effector repeats arrays were assembled according to the designed targets, and we adopted the “unit assembly” method described by Huang et al. in vitro by Luciferase SSA recombination assay in vitro Transfection Reagent . 10 ng of Renilla luciferase driven by the β–actin promoter in co-transfection with 100 ng of corresponding TALEN pair was used as the toxic reference to judge the toxicity of designed TALEN pairs. For each sample, the test and control reactions were run in triplicate. The cells were harvested 24 hours after transfection and lysed using Luciferase Cell Lysis Buffer . The relative luciferase activity was then detected by Dual-Luciferase Reporter® (DLR™) Assay System  and measured by SpectraMax L Luminescence Microplate Reader . The specific value of average firefly luciferase readout/average renilla luciferase readout (F/R) was calculated. The efficiency index was obtained by comparing the F/R value of TALEN with that of control.The efficiencies of the TALEN pairs in inducing DBS and single strand annealing (SSA) recombination were evaluated Fok I vectors were linearized with Not I  as templates, from which the capped mRNAs of TALEN pair were transcribed using mMESSAGE mMACHINE Sp6 Kit . Capped mRNAs of TALEN pair were coinjected into zebrafish embryos at the 1-cell stage.The constructed pCS2-TALE-5'-TCCTGTTCAATGTGTTTTATCAGTATGC-3' (forward) and 5'-CTTAATTTCTTCATGTTGTTCTAATGCAA-3' (reverse). PCR products were run on the agarose gel and then validated by sequencing.TALEN-injected zebrafish embryos were maintained in 0.1× Hank’s solution at 28.5°C. The survival rate was accounted at 1 day post fertilization (dpf). Genomic DNA from single 4 dpf embryos was extracted following our previously reported procedure To examine the effect of TALENs on TR modification, the length of the target TRs should be long enough to induce indels and the adjacent sequence should have suitable binding sites to design TALEN pairs. Moreover, it is better that artificial length change of the target TRs would not elicit lethal effect on the embryo.n, where n = 70) about 2.7 kbp upstream of the zebrafish ntl that has two candidate TALEN-binding sites n in both subspecies of goldfish is in accordance with that in zebrafish. Aligning comparison showed that the length of the (TG)n between zebrafish and the bisexual diploid goldfish is almost equal, but remarkably shorter (about 110bp) in the unisexual polyploid goldfish n upstream of zebrafish ntl is suitable for examining whether engineered TALENs can be employed to modify the length of TRs.By searching in the nucleotide database in EMBL, we identified a 140bp long imperfect tandem TG repeat sequence (represented with (TG)ng sites  A. Sincegoldfish  B. This n  and downstream (129bp) of the (TG)n . All theβ–actin promoter driven Renilla luciferase signal in the toxic control can be seen as an indicator of TALEN toxicity, due to off-target cleavage usually causing apoptosis of the transfected cells To determine the efficiency of the two TALEN pairs, two firefly luciferase SSA reporters were constructed by inserting the USTR TALEN or DSTR TALEN target sequences into the cloning site . BecauseLuciferase detection showed high level of Renilla luciferase signal in the URTR TALEN or DSTR TALEN pair co-tansfected control cells , suggestTo determine a appropriate dosage, different doses of the DSTR TALEN mRNA pair were microinjected into 1-cell stage zebrafish embryos separatly. At the dosage of 125 pg per embryo, the injected embryos developed as normally as the uninjected control and exhibited no specific abnormality at 1 day post fertilization . When thntl was PCR amplified (th TALEN pair targeting the tyrosine hydroxylase gene th TALEN embryos (N = 28), the length of the amplified fragment is the same as observed in wild type embryos n remained unchanged, while various indels emerged at the TALEN target site. About 5% of the sequenced clones was classified into type III. In this type sequence change occurred within both the (TG)n and the TALEN target site. Most of the examined embryos are heterozygotes or mosaics containing two or three types of clones  obtained from the heterozygotic or homozygotic mutant embryos showed that there were three types of mutation pattern  . About f clones . StrikinFok I nuclease has been widely applied in targeting genes in plant n in zebrafish genome using engineered TALENs. Our results also showed that, during the repair of the DSB induced by TALENs, mutations can occur separately within the TALEN target region or within the adjecent TR region, or simutaniusly at both regions. Therefore, all the desired mutant types of TR could be convenintly obtained by employing enginered TALENs.Since its discovery several years before, TAL effector fused with n was first discovered in the genomes from yeast to human in early 1980s by two independent groups in vitro experiments showed that the activity of chloramphenicol acetyltransferase gene was enhanced with a TG-element, and the maximum enhancement was obtained with 30–40bp of (TG)n; when the (TG)n exceeded 130bp, the gene activity declined dramatically to fivefold less compared with a 50bp (TG)nin vitro that the binding of RecA protein to (TG) n or (CA) n sequences with an increasing affinity, and the sequence recombination was promoted from 30% to 80% and 100% for DNA containing 7, 16 and 39 TG repeats n played an important role in modulating gene expression. Recent publications suggest that the loss or gain of repeats may affect the binding of the transcriptional regulatory proteins such as IHF to the promoter n region and its flanking sequence in vivo provides a strategy to explore the detailed mechanisms and the roles of (TG)n in gene expression and DNA methylation.The tandem repeat (TG)n region in zebrafish genome and elicited three types of mutations with or without TR variation. The type II mutation with indels within the TALEN target region while the (TG)n region unchanged is undoubtedly due to non-homologous end-joining  through several pathways as illustrated in Although several hypothesis, including replication slippage ng NHEJ, , which iFile S1Combined file of supporting information files. The contents include: Table S1. Primers for goldfish ntl promoter cloning. Table S2. Primers for pSSA-luciferase reporter construction. Figure S1. Alignment of upstream sequence of ntl between bisexual diploid and unisexual polyploid goldfish. Figure S2. Simplified structure of pSSA-luciferase reporter and sketch map of the SSA assay.(DOC)Click here for additional data file.


Population stratification correction resulted in 5 deletions and 3 duplications remaining significant (P = 5.16×10−5–4.26×10−2) in the replication cohort. Three deletions and four duplications were significant combined (combined P = 3.7×10−4−3.9×10−2). All associated loci were experimentally validated using qPCR. Evaluation of these genes for pathway enrichment demonstrated ∼50% are involved in alternative splicing (P = 0.0077 Benjamini and Hochberg corrected). We conclude that genetic variations disrupting RNA splicing could have long-term biological effects impacting lifespan.Longevity has a strong genetic component evidenced by family-based studies. Lipoprotein metabolism, FOXO proteins, and insulin/IGF-1 signaling pathways in model systems have shown polygenic variations predisposing to shorter lifespan. To test the hypothesis that rare variants could influence lifespan, we compared the rates of CNVs in healthy children (0–18 years of age) with individuals 67 years or older. CNVs at a significantly higher frequency in the pediatric cohort were considered risk variants impacting lifespan, while those enriched in the geriatric cohort were considered longevity protective variants. We performed a whole-genome CNV analysis on 7,313 children and 2,701 adults of European ancestry genotyped with 302,108 SNP probes. Positive findings were evaluated in an independent cohort of 2,079 pediatric and 4,692 geriatric subjects. We detected 8 deletions and 10 duplications that were enriched in the pediatric group (P = 3.33×10 The idea of extended lifespan has fascinated generations of scholarly thought. Specific diseases have been the focus of much biomedical research rather than overarching longevity which in essence successfully avoids a variety of diseases. The average lifespan of the human population has continued to increase at a slow rate due to medical and technological advances that aim at preventing and treating both acute and chronic diseases and attenuating morbidity and mortality of old age Model systems have demonstrated that lifespan can be dramatically extended by mutations in conserved pathways that regulate growth, energy metabolism, nutrition sensing, and reproduction C. Elegans with extended lifespan MINPP1 (involved in cellular proliferation) as well as LASS3 and PAPPA2 to be involved FOXO proteins IGF-1 signaling Genome instability, macromolecular aggregates, decrease in innate immunity, skin/cuticle morphology changes, decreased mitochondrial function, degenerative loss of skeletal muscle mass and strength, and decreased fitness are highly conserved phenotypes of ageing. Lifelong accumulation of various types of damage, along with random errors in DNA maintenance, might underlie intrinsic ageing. Early findings of mutant Copy number variations (CNVs) are rare losses and gains in DNA sequences that have been importantly implicated in the pathogenesis of various neurodevelopmental and psychiatric diseases The pediatric discovery group included 7,313 children recruited at the Children’s Hospital of Philadelphia . The gerTo associate CNV loci potentially contributing to shortened lifespan, we applied a segment-based scoring approach that scans the genome for consecutive probes with more frequent copy number changes in pediatric compared to geriatric subjects. The genomic span for these consecutive probes forms common copy number variation regions (CNVRs). We uncovered 101 loci with deletion and 76 with duplication enrichment in the pediatric cohort. Conversely, we identified 90 loci with deletion and 74 with duplication enrichment in the geriatric cohort . After rWe next sought to independently replicate these CNV findings in additional pediatric and geriatric subjects. CNVs were called for 2,079 young age subjects from independent pediatric cohorts all of which were recruited in the U.S.A and genotyped on the Illumina Infinium Human Hap550. We compared the CNV frequency in young with an independent cohort of 4,692 older subjects (over 50), all of which were recruited in the U.S.A. and genotyped on the Illumina Infinium Human660W-Quad. We replicated in the same direction 11 deletions and 10 duplications that were significantly enriched in the pediatric cohort, while 1 duplication was enriched in the geriatric cohort. As shown in To assess the reliability of our CNV detection method, we experimentally validated all the significant CNVRs using an independent wet lab method, quantitative real time polymerase chain reaction (qPCR)  on a ranTo fully correct for population stratification, in addition to multi-dimensional scaling, we performed principal component analysis (PCA) on the genotypes and used the resulting first three components as covariates of logistic test CNV association in the replication cohort. CNV events in our study are rare and arise randomly shown by evaluating the spatial distribution of samples having a risk CNV on the PCA plot revealing a Gaussian (at minimum uniform due to few data points) distribution which indicating minimal test statistic inflation (even less than common variants) as opposed to a small, sharply defined region Given the diverse etiology of diseases and more generally, lack of fitness in an evolutionary context, the genes underlying the broad consideration of ageing are similarly diverse. Single significant loci are certainly of interest to the common genomic CNVs resulting in specific genes to study. However, strong confidence in the result set generated can be achieved by observing the same biological system being perturbed by multiple independently significant loci. Motivated by this, genes directly overlapped by associated CNVs were prepared as a single list and non-RefSeq hypothetical gene IDs were removed. This list was entered into DAVID functional annotation enrichment tool in contrast with a background representing genome-wide regions covered by the array. Taking into account the size of different genes and the gene family size of different annotations, the enrichment of our CNV impacted list was assigned a p-value with Benjamini and Hochberg correction for multiple testing. Functional annotations from multiple databases were used including KEGG and GO (gene ontology). Functional categories were reviewed for genes contributing from distinct genomic regions to reject enrichment of closely clustered gene families.AGPAT3, BTBD12, NLRC3, RECQL5, SCAPER, ACOT7, C19orf62, C21orf33, C22orf25, ESPN, HES2, LUZP2, NFIC, OBSCN, PDE4A, PLEKHG5, PLXDC1, KCNT1, PDXK, RAPSN, RRP1B, RNF212, SGSM2, SLC38A10, SLC39A13, and TNFRSF25 all of which were significantly enriched in the young age group (P = 0.0077 Benjamini and Hochberg corrected), suggesting that genetic variations that disrupt RNA splicing may have long-term biological effects on human lifespan.To identify potential functional biases specific to CNVs observed at significantly higher frequency in young individuals, we evaluated clustering into specific functional categories using DAVID Limited nutrition, somatic maintenance and growth are pathways to longevity. Emphasis on somatic maintenance is more important than early growth and reproduction. Post-transcriptional modification of mRNA is an important mechanism which results in a variety of protein isoforms and occurs in at least 80% of human genes, and known to harbor variations that have been associated with human disease Alternative splicing is an abundant violation of the one gene one protein theory initially regarded. The exons of an mRNA can be edited producing a variety of combinations which result in a variety of protein isoforms. This mechanism allows for a great diversity of protein products based on the same DNA code and branches out gene families much the way ancestral duplications extends gene families in DNA. Proteins responsible for alternative splicing bind to specific RNA sequences to promote or repress splicing.ADARB1 and ADARB2 were associated with extreme old age in a United States based study of centenarians with replication to four other ethnic backgrounds BTBD12 and BABAM1 are part of a multi-protein complex containing enzymes involved in DNA maintenance and repair of serious damage such as collapsed replication forks and double-strand breaks (DSBs) BABAM1 is the most highly significant CNV associated locus following full statistical correction of population stratification (p = 5.16×10−5).SNPs in the RNA editing genes ACOT7 is involved with biosynthesis of unsaturated fatty acids and decreased expression is associated with mesial temporal lobe epilepsy. Young individuals showed significantly higher frequency of both deletions and duplications of this locus compared to older individuals , recruited for the AGES-Reykjavik study We performed high-throughput, genome-wide SNP genotyping, using the InfiniumII HumanHap550 BeadChip technology (Illumina San Diego CA), at the Center for Applied Genomics at CHOP. The genotype data content together with the intensity data provided by the genotyping array provides high confidence for CNV calls. Importantly, the simultaneous analysis of intensity data and genotype data in the same experimental setting establishes a highly accurate definition for normal diploid states and any deviation thereof. To call CNVs, we used the PennCNV algorithm, which combines multiple sources of information, including Log R Ratio (LRR) and B Allele Frequency (BAF) at each SNP marker, along with SNP spacing, a trained hidden Markov model, and population frequency of the B allele to generate CNV calls. The intersection set of 302,108 probes common to the Illumina 550K: 532,898 probes and Illumina 370 Duo: 370,405 probes was used to make datasets as comparable as possible.We calculated Quality Control (QC) measures on our HumanHap660 GWAS data based on statistical distributions to exclude poor quality DNA samples and false positive CNVs. The first threshold is the percentage of attempted SNPs which were successfully genotyped. Only samples with call rate >98% were included. The genome wide intensity signal must have as little noise as possible. Only samples with the standard deviation (SD) of normalized intensity (LRR) <0.30 were included. All samples must have clear European ethnicity based on Eigenstrat smartPCA scoring and all other samples were excluded. Wave artifacts roughly correlating with GC content resulting from hybridization bias of low full length DNA quantity are known to interfere with accurate inference of copy number variations. Only samples where the GC wave factor of LRR |GCWF|<0.05 were accepted. If the count of CNV calls made by PennCNV exceeds 100, the DNA quality is usually poor. Thus, only samples with CNV call count <100 were included. Any duplicate samples (such as monozygotic twins) had one sample excluded.CNV frequency between cases and controls was evaluated at each SNP using Fisher’s exact test. We only considered loci that were significant between cases and controls (p<0.05) where cases in the discovery cohort had the same variation, replicated in an independent cohort or were not observed in any of the control subjects, and validated with an independent method. We report statistical  local minimums to narrow the association in reference to a region of nominal significance including SNPs residing within 1 Mb of each other. Resulting significant CNVRs were excluded if they met any of the following criteria: i) residing on telomere or centromere proximal cytobands; ii) arising in a “peninsula” of common CNV arising from variation in boundary truncation of CNV calling; iii) genomic regions with extremes in GC content which produces hybridization bias; or iv) samples contributing to multiple CNVRs. A peninsula is defined as a false positive association arising from a region of common CNV extending variably due to variability in probe performance and variability in samples. In other words, the specific significant subregion is confounded by contributing calls also extending to a non-significant subregion.To fully correct for population stratification, we performed (PCA) on the genotypes and used the resulting first three components as covariates of the logistic test for CNV association using Plink.ip is the p-value for the ith study. Under the null hypothesis, X2 follows a chi-squared distribution with 2k degrees of freedom, where k is the number of studies. In this case, there were two studies yielding a chi-squared distribution with four degrees of freedom.Combined p-values were calculated using Fisher’s methodTo inform multiple testing correction, CNV filtering steps have been performed as part of the analysis. Firstly, it is important to note that of the intersection set of 302,108 SNPs on the Illumina array, 3,911 (1.295%) showed deletion and 8,830 (2.923%) showed duplication in at least eleven or more unrelated cases in the discovery cohort (frequency ≥0.150%). 41,392 (13.701%) deletion and 45,050 (14.912%) duplication SNPs were observed in at least two individuals. The threshold of three cases harboring a given CNV is selected because it is the minimal case frequency to provide minimal expectation of frequency differences between cases and controls to yield nominal statistical significance and reproducibility for the calls in a given region. We find this upfront exclusion to be very similar to the inclusion threshold of 1% minor allele frequency in GWA SNP genotype studies. These SNPs were collapsed into 101 deletion and 76 duplication CNVRs based on necessary multiple neighboring SNP signals to call a CNV and resulting redundancy of individual SNP statistics. This results in a total of 171 tests being performed corresponding to a multiple testing correction bar of p = 2.92E-4 close to the p = 5E-4 bar we have seen previously.Given the diverse etiology of diseases and more generally, lack of fitness in an evolutionary context, the genes underlying the broad consideration of ageing are similarly diverse. Single significant loci are certainly of interest to the common genomic CNVs resulting in specific genes to study. However, strong confidence in the result set generated can be achieved by observing the same biological system being perturbed by multiple independently significant loci. Motivated by this, genes directly overlapped by associated CNVs were prepared as a single list and non-RefSeq hypothetical gene IDs were removed. This list was entered into DAVID functional annotation enrichment tool in contrast with a background representing genome-wide regions covered by the array. Taking into account the size of different genes and the gene family size of different annotations, the enrichment of our CNV impacted list was assigned a p-value with Benjamini and Hochberg correction for multiple testing. Functional annotations from multiple databases were used including KEGG and GO (gene ontology). Functional categories were reviewed for genes contributing from distinct genomic regions to reject enrichment of closely clustered gene families.


MMTV is a type B betaretrovirus which preassembles intracellularly, a phenomenon distinct from retroviruses that assemble the progeny virion at cell surface just before budding such as the type C human and feline immunodeficiency viruses (HIV and FIV). Studies of FIV and Mason-Pfizer monkey virus (MPMV), a type D betaretrovirus with similar intracellular virion assembly processes as MMTV, have shown that the 5′ untranslated region (5′ UTR) and 5′ end of gag or 5′ UTR sequences, or incremental amounts of 5′ UTR in the presence of 400 nucleotides (nt) of gag were constructed to delineate the extent of 5′ sequences that may be involved in MMTV gRNA packaging. Real time PCR measured the packaging efficiency of these vector RNAs into MMTV particles generated by co-transfection of MMTV Gag/Pol, vesicular stomatitis virus envelope glycoprotein (VSV-G Env), and individual transfer vectors into human 293T cells. Transfer vector RNA propagation was monitored by measuring transduction of target HeLaT4 cells following infection with viral particles containing a hygromycin resistance gene expression cassette on the packaged RNA.Three series of MMTV transfer vectors containing incremental amounts of gag for not only efficient gRNA packaging but also propagation of MMTV-based transfer vector RNAs. Vector RNAs without the entire 5′ UTR were defective for both efficient packaging and propagation into target cells.MMTV requires the entire 5′ UTR and a minimum of ∼120 nucleotide (nt) at the 5′ end of These results reveal that the 5′ end of MMTV genome is critical for both gRNA packaging and propagation, unlike the recently delineated FIV and MPMV packaging determinants that have been shown to be of bipartite nature. However, early MMTV studies showed that MMTV genome containing deletion in the gag sequences important in these processes. Using several series of MMTV sub-genomic vectors in a biologically relevant in vivo packaging and transduction assay, our results reveal that unlike retroviruses such as FIV gag, for efficient gRNA packaging and propagation of the packaged transfer vector RNA into the infected cells.In order to further map the minimum viral sequences important for MMTV gRNA packaging and transfer vector RNA propagation, a systematic deletion approach was employed to delineate the extent of 5′ UTR and in vivo RNA packaging and propagation assay for MMTV was employed that utilizes an MMTV gag/pol expression construct (JA10) for the production of viral structural and enzymatic proteins To study MMTV RNA packaging, the newly developed hygromycin resistance gene driven from the simian virus 40 (SV40) promoter as a marker for successful propagation, a truncated MMTV env gene, and the 3′ MMTV LTR  even with the vector DA07 containing the entire 5′ UTR and 400 nt of gag  that could be propagated, although inefficiently, was tested. Reverse transcriptase (RT) PCR of the DA series of vector RNAs packaged by the virus particles revealed that the vector RNAs could be packaged, but quite inefficiently with some of the samples requiring Southern blotting for visualization of the packaged RNA  regulatory export pathway. This pathway has recently been described in MMTV To circumvent any potential problems with poor MMTV promoter efficiency in human cells and RNA export defects, the 5′ MMTV LTR was modified by replacing the U3 region (containing the promoter) with the constitutively active human cytomegalovirus (hCMV) promoter that has been classically used as a high efficiency promoter in mammalian cells . A similenv expression vector MD.G for virus production into 293T cells. Following transfection, virus particles were isolated from each transfected cultures to quantify the effect of the introduced mutations on both gRNA packaging and propagation. However, before quantifying the viral RNA contents in the viral particles, it was imperative to determine whether each vector RNA was expressed stably and transported to the cytoplasm successfully.The modified transfer vectors  were co-In order to ascertain stable expression and efficient nuclear export of transfer vector RNAs, nuclear and cytoplasmic RNA fractions were prepared from the transfected cells and DNase-treated to ensure removal of any contaminating plasmid DNA ; panel IThe relative expression of the various transfer vector RNAs in the cytoplasm was tested using a custom-made MMTV Taqman real time PCR assay in combination with a commercially-available-endogenous β-actin Taqman assay (Applied Biosystems). The custom MMTV Taqman assay was designed within the U5 region of the transfer vector RNAs, enabling detection of all mutant transfer vector RNAs via a single primer/probe combination. To allow a relative comparison of the transfer vector RNA expression using β-actin as an endogenous control, we determined whether the amplification efficiencies of the two assays were equivalent. This was accomplished by first determining the value of the threshold cycle, Ct , followeTest of the cDNAs prepared from the cytoplasmic RNA fractions revealed that the transfer vector RNAs were stably expressed between Ct values of 23.5–27.5 and efficiently exported to the cytoplasm . AlongsiNext, the packaging efficiency of the transfer vector RNAs into the pseudotyped MMTV particles was assessed by the same custom-made real time PCR assay as described above. Towards this end, the first question asked was whether a cellular housekeeping β-actin mRNA could be used as an endogenous control in our assays since it is not a recognized virion-associated mRNA. It has now been well-established that cellular RNAs can be packaged into retroviral particles as has been shown for several RNAs such as 7SL RNA, U6 snRNA, tRNA, or mRNAs for cyclophilin A and for several types of ribosomal proteins, and even ribosomal RNA itself As opposed to β-actin mRNA, all transfer vector RNAs were encapsidated with widely different packaging efficiencies, encompassing raw Ct values between 23–36, depending upon the amount and type of sequence present at the 5′ end of the sub-genomic RNAs (see boxed region in 10 Relative Quantification (RQ) units (∼ three-folds above the mock level).Further normalization of the transfer vector RNAs packaged into the virus particles was performed to take into account differences in transfection efficiency observed using the luciferase expression vector in each transfected cultures . The samgag were the most efficiently packaged  compared to vector series with incremental amounts of 5′ UTR either without gag (FA series) or with gag (NS series) except for NS12 (gag) that was efficiently packaged and comparable to the DA series of vectors. Overall, these results revealed that the intact 5′ UTR is critical for efficient RNA packaging but is not sufficient by itself for this crucial step of MMTV life cycle.Normalization of the data revealed that the DA series of transfer vectors with the entire 5′ UTR and incremental amounts of for NS12 . In factfor NS12 , packagigag sequences increased, so did the packaging efficiency with DA24 containing 400 nt gag being the vector packaged most efficiently (gag respectively and these vector RNAs were packaged within < one log of DA24 (p<0.01). These results corroborated well the observations made earlier using MMTV vectors harboring similar sequences and containing wild type MMTV LTR as the promoter , as the incremental amount of iciently . Howeverpromoter  that 120 compare  and 5C.gag (comparable to DA24), showed lower packaging efficiency  and was similar to DA22 and DA23, vectors with 120 and 150 nt of gag sequences, respectively  resulted in greater than 100-fold (∼2 log) drop in RNA packaging efficiency compared to the vector RNA with the entire 5′ UTR, NS12 (gag (FA26) was packaged at a greater than 10-fold (1 log) lower efficiency than NS12 and greater than 60-fold (∼1.8 log) lower efficiency than DA24, vectors containing the entire 5′ UTR in the presence of 400 nt of gag (p<0.01), confirming that gag sequences are indeed required during MMTV gRNA packaging  contains elements critical for proper completion of steps in vector RNA propagation, such as RNA dimerization, encapsidation, reverse transcription, and integration since our readout assay for RNA propagation was dependent on the successful completion of these crucial steps in virus life cycle, and 2) the entire 5′ UTR itself is not sufficient and requires ∼120–400 nt of gag for efficient vector RNA propagation. Overall, these results mimicked the RNA packaging and propagation results obtained with the wild type MMTV LTR vectors and further validated that the intact 5′ UTR is as critical for efficient RNA propagation as it is for successful RNA packaging  LTR gag sequences, 2) the insensitivity of the assays used to study RNA packaging. In their assay, RNA from a 100 ml of a wild type virus stock containing 2×107 virions/ml  was required to observe packaging efficiently on a dot blot assay. In contrast, in our real time-based PCR assay, we could efficiently observe packaged retroviral subgenomic RNAs from vectors that gave rise to even 25 CFU/ml   or provide stability to the RNA higher order structure that the 5′ region may assume. Consistent with this, it has recently been shown that at least for FIV and MPMV, the gag sequences are involved in long range interaction (LRI) with sequences at the 5′ end of the gRNA gag sequences results in destabilization of the overall RNA secondary structure and consequently, RNA packaging and propagation gag sequences is not so clear in the case of SIV since most of the studies conducted on SIV RNA packaging have maintained gag sequences gag region (9 nts) may be required for efficient RNA packaging without affecting RNA dimerization or nucleocapsid binding Spe I restriction site insertion between the end of 5′ UTR and gag AUG had resulted in lower packaging efficiency of the NS12 vector compared to the DA24  . Considethe DA24 .Over the years, it is becoming increasingly clear that retroviral RNA packaging and dimerization, regardless of the primary sequence, are dependent on RNA structural motifs of the packaging determinants and their interaction(s) at the structural level (RNA-RNA interactions during dimerization and RNA-protein interactions during packaging) (reviewed in gag/pol genes in the presence of the MPMV CTE using the hCMV promoter and the bovine growth hormone (BGH) poly A sequences. The transfer vectors were created as three series: the DA series with incremental deletions of gag sequences in the presence of the entire 5′ UTR, the FA series with incremental deletions of the 5′ UTR sequences in the absence of gag, and the NS series with incremental deletions of the 5′ UTR sequences in the presence of 400 nt of gag  by the pseudotyped virus particles and selection for hygromycin-resistant colonies, as described previously The human epithelial kidney cell line, 293T, was used for the production of VSV-G-pseudotyped-MMTV virus particles containing RNA from the various constructs using a modified calcium phosphate method as described Transfected cells were fractionated into nuclear and cytoplasmic fractions to allow separate analysis of RNA residing within the two cellular compartments as described earlier GTC CTA ATA TTC ACG TCT CGT GTG, nt 1179–1202 and OTR 672 (−) CTG TTC GGG CGC CAG CTG CCG CAG, nt 1298–1321. Hybridization was performed using the AlkPhos Direct Labeling Kit  as per manufacturer's directions.Some of the packaged viral RNA samples were subjected to Southern blot analysis as described earlier TCGCCATCCCGTCTCC ; Forward primer (MTV-1LTR-SITEF): CGTCTCGTGTGTTTGTGTTTGTGTCTGT ; Reverse primer (MTV-1LTR-SITER): CCTCTGGAAAGTGAAGGATAAGTGA . The assay was validated in silico using the Primer Express tool as well as the ABI design pipeline bioinformatic tools that ensure specificity, reproducibility, and amplification efficiency of the assay performance. Further validation of the PCR amplification efficiency was conducted by testing the assay using the online web tool “pcrEfficiency” To test the relative levels of transfer vector RNAs expressed within the cytoplasm and packaged into the viral particles, a real time PCR custom expression assay was developed using the minor groove binding (MGB)/FAM and MGB/VIC chemistry from Applied Biosystems, USA. Additionally, the ABI real time PCR assays use ROX as an internal fluorescence reference dye to which the target reporter dye signal  is normalized to during data analysis, to correct for fluorescence fluctuations caused by changes in concentration or volume. The PCR primers were designed to anneal within a region at the beginning of the MMTV U5 region (nt 1192–1259), a 68 nt region common to all the constructs, enabling their relative quantitative assessment using the same probe/primer combination concomitantly. The sequence of the probe and primers were as follows: Probe (MTV-1LTR-SITEM1 FAM): To determine which method to use for the Relative Quantification analysis, the standard curve or the comparative (ΔΔCt) method, a 10- and serial 2-fold dilutions of a cDNA prepared from 293T cells expressing DA24 were tested in non-multiplex RT-PCRs in triplicates assuming conservatively that 1 ug of input RNA results in ∼100 ng of single stranded cDNA using random hexamers as primers and MMLV RT Promega; . The resth of the total viral RNA preparations were first DNase-treated, confirmed for the absence of DNA by PCR using vector RNA specific primers, and then reverse transcribed into cDNA, as previously described For the final relative quantification analysis, each viral RNA sample was tested in triplicates in multiple assays using the Taqman Universal Master Mix , while the cytoplasmic RNA was tested in duplicates. As mentioned earlier for the cytoplasmic RNA fractions, 1/5ttest was performed between various constructs using the Microsoft Excel software. A value of p<0.01 was considered statistically significant.To determine whether the observed differences in the normalized packaging efficiencies were statistically significant, a standard, paired, two-tailed Student's


Magnetic resonance diffusion tensor imaging, which is based on the measuring the directional diffusivity of water molecules, is an established method of evaluating tissue structure. Prolonged imaging times have precluded the use of embryonic diffusion tensor imaging due to motion artifact. Using temperature-based motion suppression, we aimed to investigate whether diffusion tensor imaging can be used to monitor embryonic smooth muscle development From a set of 82 normally developing fertile chicken eggs, 5 eggs were randomly chosen each day from incubation days 5 to 18 and cooled using a dual-cooling technique prior to and during magnetic resonance imaging at 3.0 Tesla. Smooth muscle fibers of the gizzard were tracked using region of interests placed over the gizzard. Following imaging, the egg was cracked and the embryo was fixated and sectioned, and a micrograph most closely corresponding to the acquired magnetic resonance image was made. Smooth muscle fiber fraction was determined using an automated computer algorithm.in ovo development of smooth muscle tissue.We show that diffusion tensor images of smooth muscle within the embryonic gizzard can be acquired in ovo from incubation day 11 through hatching. Length of tracked fibers and day of incubation were found to have statistical significance (p<0.05) by multiple linear regression correlation with histologic specimens of sacrificed embryos from day 11 of incubation through hatching. The morphologic pattern of development in our histologic specimens corresponds to the development of embryonic gizzard as reported in the literature. These results suggest that diffusion tensor imaging can provide a noninvasive method of evaluating  In vivo evaluation of tissue structure improves the understanding of tissue differentiation within the developmental environment in vivo anatomic imaging, and has been used to image avian embryos. With the refinement of advanced MR techniques such as magnetic resonance spectroscopy, blood-oxygen level dependent imaging, and diffusion weighted imaging, anatomic data can be augmented with metabolic, physiologic, and structural information in ovo anatomic images of chick embryos Animal models are essential for understanding embryonic development and disease pathogenesis from the anatomic to the molecular level. in ovo using embryonic cooling, and investigate whether serial DTI smooth muscle tractography correlates with ex vivo histology.Harnessing the effects of Brownian motion, diffusion weighted imaging (DWI) translates the restrictive effect of tissue structure on water molecule mobility into visible signal intensity differences The proventriculus (primitive gizzard) could be discriminated on T2 weighted images from day 7. Beginning with day 11, muscle fibers of the gizzard could be tracked with DTI and the fiber length derived. As shown in 1(Day)-0.04275 X2(FA)-0.01869 X3(Length)  and the multiple correlation coefficient is 0.9688. Typical examples of DTI derived serial smooth muscle fiber tracking result are shown in While fractional anisotropy values could be calculated via tissue diffusion measurements as early as day 7, diffusion-tensor tracking of muscle fibers was not successful until incubation day 11, which is essentially the same time that muscle fibers could be visualized on histologic specimens (days 10–11). Multiple linear regression analysis of FiberArea%, day of incubation (Day), smooth muscle fractional anisotropy (FA), fiber tract length (Length) from incubation days 11 through 18 is given in ex vivo analytical methods, which typically involve embryonic sacrifice, repeat observations can be performed on a single embryo throughout the incubation period.Our data show that muscle fiber tracts can be discerned during the incubation period on diffusion tensor images when motion is sufficiently suppressed by cooling prior to and during imaging. In contrast to Smooth muscle development of chick embryo gizzard has been previously elucidated We were able to track smooth muscle fibers within the gizzard using diffusion tensor imaging on day 11, consistent with our histologic findings and findings discussed in the literature. Progressive fiber length elongation correlates with our histological quantification by multiple linear regression analysis, and shows diffusion fiber tracking in DTI to be a noninvasive method of monitoring smooth muscle fiber development. FA shows the degree of anisotropy . Our histological analysis method (FiberArea%) depends on the enlargement of trellis-like muscle bundle separated by the collagen bands, and there is no linear correlation between muscle bundle enlargement and myoblast/myofiber anisotropy in our multiple regression analysis. Small samples (only 5 eggs for each day) and selected bias  may be a limitation and influence the results.in ovo by diffusion tensor imaging. Diffusion tensor magnetic resonance imaging has been shown to reveal regional dysmyelination effects in the Shiverer mouse mutant, with diffusion-tensor acquired parameters correlating with histologic microanatomic changes in vivo diffusion tensor imaging parameters correlating with histologic demyelination of injured regions We believe that this method has the potential to allow noninvasive evaluation of other tract-like organizing tissue within avian embryos such as nerve fibers, though the number of diffusion directions would be increased  and the subsequent increase in imaging time due the increase in the number directions while maintaining isotropic voxels would necessitate the use of a dedicated animal magnet. It is possible that developmental mutational abnormalities or injuries could be detected in vivo imaging and ex vivo histology, acquiring exact matches between imaging planes and histologic specimens is technically challenging. Even with careful attention to detail, exact matching was not feasible in all cases. Chick embryo orientation and position within the egg naturally changes between successive days of imaging, and limits the ability to exactly match imaging planes between successive days. Despite this limitation, the proventriculus or gizzard could be consistently identified on magnetic resonance images after day 6. Lastly, while not apparent in our small sample, developmental differences may exist between embryos of the same hatching day.This study has several inherent limitations. Small embryonic structures, primarily during the early period of incubation, are difficult or impossible to evaluate due to the spatial resolution limitations of 3.0 T MRI; higher field strength magnets should improve spatial resolution. As in any study correlating in vivo evaluation of embryonic development. We show that tracked fiber length of smooth muscle in diffusion tensor imaging correlates with parameters derived from histologic specimens from sacrificed embryos. Noninvasive evaluation of development within embryonic models has predominantly consisted of anatomic evaluation. Diffusion tensor magnetic resonance imaging could provide a new method of understanding the mechanics of early development.Our study shows that diffusion tensor imaging of smooth muscle, using a widely-available 3.0 T magnetic resonance imaging system, holds promise as an investigative method for the serial The experimental protocol and procedures were approved by the Institutional Ethics Committee of the Shanghai Jiaotong University School of Medicine. Ninety (90) fertile Hy-Line White eggs each weighing 50–55 g were obtained from a commercial hatchery and placed in a digital tabletop incubator with temperature (37.8°C) and humidity (60%) controlled automatically. After four days of incubation, embryo development was evaluated by candling  to determine if they were fertile and developing normally. Eight eggs were removed from the incubator for underdevelopment. From the 82 remaining eggs, 5 eggs were chosen at random each day, from incubation days 5 to 18  and treated as follows: eggs were removed from the incubator and air-cooled for one hour at 3.5–4°C prior to imaging th unweighted (b = 0) DTI image was also acquired. Eggs wrapped in Techni-Ice were imaged in a 3.0 T Philips Achieva magnet  using a four-channel dedicated animal coil with a 5 cm inner diameter. Image acquisition consisted of high-resolution T2 weighted TSE (turbo spin echo) images , slice thickness 1.2 mm, no gap, 12 min 23 s duration), and SENSE DTI using SE (spin echo)-DWI , b = 500 s/mm2, NEX = 2, slice thickness 1.2 mm, no gap, 20 minute duration). All imaging planes were sagittal. A 16Smooth muscle fibers of the gizzard were tracked using FiberTrak software . Diffusion registration was used prior to fiber tracking. Three parameters, minimum fractional anisotropy (FA), maximum angle change, and minimum fiber length were set to determine the stopping threshold of tractography. We chose a small minimum FA (0.15) and small maximum angle change (27°) to balance the sensitivity of smooth muscle fibers' tracking during early development stage and the precision during later development. A small minimum fiber length (5 mm) was used to enhance sensitivity during early development, with a longer minimum fiber length (10 mm) used during later development to avoid unrelated fibers from being tracked. High-resolution T2-weighted images were used to guide placement of the region of interest (ROI) required for seeding the fiber tracking algorithm. The sagittal image with the largest section of the gizzard was chosen for ROI placement. The ROI size changed with embryonic development; prior to day 11 of incubation, the ROI covered the entire proventriculus. After day 11 of incubation, two ROIs were used, each covering about half of the gizzard. Example ROIs are shown in After MR imaging, the egg was cracked and the corresponding embryonic proventriculus or gizzard was resected as a specimen and fixed in a 10% formaldehyde solution for one week. Following dehydration and paraffin embedding, the specimen was serially sectioned at 3 µm, in a plane corresponding to the sagittal MR imaging plane; sections were then stained with hematoxylin and eosin. Each specimen was evaluated with light microscopy . The block region most closely corresponding to the DTI ROI was chosen and micrographed  value of tissue within ROI, an invariant scalar index which accounts for structural anisotropy in tissue, was automatically computed by the FiberTrak software. FA is defined as:Both statistical analysis and data management was performed using MedCalc . A linear model was used to evaluate the relationship between day of incubation and ROI-derived fractional anisotropy (FA) values, Length of tracked fibers, and FiberArea%. To evaluate the relationship between imaging and histology during embryonic development, the multiple regression analysis among FiberArea% (dependent), day of incubation (independent), FA of ROI (independent) and Length of tracked fibers (independent) was performed, with p values = 0.05 considered to be significant.