Caenorhabditis elegans HNF4α-related nuclear hormone receptor NHR-62 is required for metabolic and physiologic responses associated with DR-induced longevity. nhr-62 mediates the longevity of eat-2 mutants, a genetic mimetic of dietary restriction, and blunts the longevity response of DR induced by bacterial food dilution at low nutrient levels. Metabolic changes associated with DR, including decreased Oil Red O staining, decreased triglyceride levels, and increased autophagy are partly reversed by mutation of nhr-62. Additionally, the DR fatty acid profile is altered in nhr-62 mutants. Expression profiles reveal that several hundred genes induced by DR depend on the activity of NHR-62, including a putative lipase required for the DR response. This study provides critical evidence of nuclear hormone receptor regulation of the DR longevity response, suggesting hormonal and metabolic control of life span.Dietary restriction (DR) extends lifespan in a wide variety of species, yet the underlying mechanisms are not well understood. Here we show that the  Caenorhabditis elegans, genes implicated in the TOR pathway, autophagy, protein synthesis and energy homeostasis have been shown to modulate the dietary restriction response; however their mechanism of action is still unclear. In this work, we find that the C. elegans nuclear hormone receptor, nhr-62, is required for longevity in multiple DR regimens, providing the first evidence of a nuclear receptor required for DR-induced longevity. Additionally, nhr-62 is required for physiologic changes associated with DR, including increased autophagy and decreased levels of triglycerides, possibly through lipolysis. Moreover, nhr-62 is responsible for regulating hundreds of genes under DR, as measured by qPCR and RNA-seq. Importantly, this work is the first to report transcriptome analysis of DR in C. elegans and the first to provide functional evidence that nuclear receptors are key regulators of the DR longevity response, which imply hormonal and metabolic control of longevity, possibly through alterations in fat metabolism, lipolysis, and autophagy.Dietary restriction extends the life span of diverse species across taxa, yet the underlying mechanisms are poorly understood. In humans there are clear health benefits associated with DR such as improved serum cholesterol and lipid levels. In  Genetic and environmental factors can cause profound changes in organism lifespan. Genetic alterations that stimulate robust longevity across taxa include reduced insulin/IGF and TOR signaling, reduced mitochondrial function, and reduced signaling from germline stem cells Caenorhabditis elegans several different DR regimens can induce longevity. The two most widely used are dilution of bacterial food and the genetic DR mimetic eat-2. Dilution of bacterial food in liquid culture (BDR) was first demonstrated by Klass in 1977 to extend C. elegans life span, and variations of this method have been shown to enhance longevity by 20–100% ad libitum until adulthood, and then are shifted to liquid culture containing a dilution of bacterial food. The eat-2 mutation affects the function of a pharyngeal acetylcholine receptor subunit, which reduces pharyngeal pumping rate and subsequent food intake throughout the life of the animal, and extends life span by 15–40% C. elegans adults include intermittent feeding (IF), in which worms are fed every two days, dietary deprivation (DD), in which adult worms are completely removed from food, and solid DR (sDR) where bacteria is diluted on solid agar plates In C. elegans, a few key transcriptional regulators of the DR response have started to emerge. PHA-4 is a FOXA homolog required for eat-2 and BDR induced longevity From genetic studies in nhr-62 as required for DR-induced metabolic and longevity responses.In an effort to identify regulators of DR-induced longevity, we specifically focused on nuclear hormone receptors (NHRs). Nuclear hormone receptors are transcription factors that respond to fat-soluble hormones, such as steroids and fatty acids, to directly regulate gene transcription. They are broadly implicated in the regulation of development, metabolism and homeostasis, and are well poised to coordinate events throughout the body in response to hormonal or nutritional signals eat-2 mutation (a genetic DR mimetic), and screened for a loss of eat-2-induced longevity. We screened 246 of the 284 C. elegans NHRs in a genetic background more sensitive to RNAi . Knockdown of most NHR genes had no effect on eat-2 longevity  yet largely maintained DR-induced life span extension  consisting of DNA- and ligand-binding domains , and a short isoform B (353 AA) consisting of only the LBD , which removes a 658 bp region, including part of exon one, and results in an immediate stop codon, thus removing the DBD of the receptor. Although nhr-62(tm1818) is likely a strong loss-of-function allele, it may not be null because the predicted B isoform containing the LBD only is intact. We introduced this mutation into eat-2(ad465) animals and measured the lifespan of the double mutant. Similar to RNAi, nhr-62(tm1818) significantly suppressed the lifespan of eat-2(ad465) animals. Importantly, the nhr-62(tm1818) mutant had little or no effect on wild-type (N2), suggesting that nhr-62 does not suppress eat-2 longevity through general sickness (nhr-62(tm1818) consistently suppressed eat-2(ad465) lifespan, suppression was not always complete, suggesting other activities still contribute to the response.To validate our RNAi results, we obtained the deletion allele, sickness . Though nhr-62 in DR-induced longevity, we generated an extra chromosomal line, dhEx627, expressing wild-type nhr-62, and introduced it into eat-2;nhr-62 mutant animals to measure whether complementing nhr-62 function was sufficient to restore longevity. As expected, the dhEx627 array restored longevity to eat-2;nhr-62 double mutants  without affecting pharyngeal pumping rate  animals fed daf-2 (IR) or cco-1 (cytochrome c oxidase) RNAi. As expected, daf-2 RNAi and cco-1 RNAi robustly increased the life span of wild-type animals. Moreover, RNAi knockdown similarly increased longevity in the nhr-62(tm1818) background  signaling or reduced mitochondrial function have both been shown to extend life span ckground . These rnhr-62(tm1818) is also required for DR-induced longevity by BDR, we measured the lifespan of wild-type and nhr-62(tm1818) worms fed bacteria at ten different concentrations. We started with our ad libitum bacterial concentration of 2.5×109 CFU/ml followed by nine subsequent two-fold serial dilutions. At the level of DR associated with the longest lifespan , wild-type worms exhibited a 188% increase in median lifespan compared to ad libitum conditions (nhr-62(tm1818) worms exhibited a 40% increase in median lifespan compared to ad libitum conditions, identical to the wild-type DR response curve at these high nutrient conditions. However, at the optimal DR condition, the DR response curve for nhr-62(tm1818) mutants diverged from the wild-type curve; these animals were unable to respond to DR as effectively, with only a partial increase in the median lifespan at all subsequent concentrations . As previously described, transgene expression was observed from embryo to adult in the pharynx and intestine. We also observed clear expression in various neurons including sensory neurons, motor neurons, hermaphrodite specific neuron, and pharyngeal neurons (data not shown). A similar pattern of expression was seen in the eat-2(ad465) background. A full-length integrated low copy nhr-62::gfp expressing strain from the TransgeneOme Project eat-2 backgrounds. Again no obvious change in nhr-62::gfp intensity or localization was observed in the eat-2 background, nor were changes seen in nhr-62 mRNA levels as measured by qPCR. Notably several tissues that express nhr-62::gfp, such as the intestine, ASI sensory neurons, and hypodermis, are major endocrine tissues that coordinate metabolic states and have been implicated in aging We obtained a transgenic strain from the Hope laboratory containing 2 kb of the ad libitum conditions. Since nhr-62 shares homology to HNF4α-like NHRs known to function in fat metabolism such as nhr-49 and nhr-80nhr-62 might be necessary for proper fat regulation under DR. To test this hypothesis, we treated wild-type, nhr-62(tm1818), eat-2(ad465), and eat-2;nhr-62 animals with the lysochrome dye Oil Red O, which stains neutral triglycerides and lipids  nhr-62 regulates fatty acid composition under DR, we quantified individual fats by gas chromatography. eat-2;nhr-62 double mutants exhibited increased levels of saturated fatty acids compared to wild-type. Moreover, eat-2;nhr-62 double mutants showed a significant reduction of various monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) compared to eat-2 mutants alone , a nucleophilic elbow, and other features typical of lipases  mutants. We found that let-363 knockdown induced longevity in both wild-type and nhr-62 mutants  kinase. Down-regulation of  mutants . These rnhr-62 promotes DR longevity and associated physiological processes, we speculated that it could modulate the overall transcriptional change upon DR. To test this hypothesis, we performed RNA-seq on wild-type, nhr-62(tm1818), eat-2(ad465), and eat-2;nhr-62 animals. This approach not only enabled us to identify genes regulated under DR, but also which genes showed nhr-62 dependence. Our criteria for differential gene regulation included a greater than 1.5-fold change in expression compared to wild-type, a false discovery rate less than 0.05, and greater than 10 reads when normalized to the base mean. For each sample we typically obtained 25–37 million unambiguous reads, and could assign these to approximately 15,000 genes. Heat maps of the mean Euclidean distance between samples showed that nhr-62 was most similar to wild-type, while eat-2 was most different, with eat-2;nhr-62 in between  back to wild-type levels , phosphorus metabolism , nucleosome assembly and organization,  protein, amino acid, histidine, and heterocyclic amine metabolism  and short chain dehydrogenase metabolism   ,S7. OtheC. elegans we identified the HNF4α homolog, nhr-62, as an important mediator of the DR response. Several lines of evidence are consistent with the notion that nhr-62 promotes aspects of DR-induced metabolism and longevity. RNAi knockdown or mutation of nhr-62 specifically suppressed eat-2 DR-induced longevity, but not that of reduced insulin/IGF receptor (daf-2) or mitochondrial function (cco-1) arguing for a DR specific function. Importantly, nhr-62 mutation prevented animals from fully responding to the longevity-inducing effects of bacterial food dilution (BDR), thereby making it one of only a handful of DR-regulating genes that clearly modulates multiple forms of DR. By comparing eat-2 to eat-2;nhr-62 mutants we found a reversal of Oil Red O staining intensity, triglyceride levels, and autophagy induction, without seeing a difference in body size or pumping rate. Additionally, we found that nhr-62 mutation decreased the mono- and polyunsaturated fatty acid content of dietary restricted worms. Finally, we found a reversal of the DR-induced transcriptional regulation of a predicted lipase required for DR, as well as a plethora of genes through RNA-seq analysis. These results point to a model in which a dietary restricted state promotes activation of nhr-62, the lipase C40H1.8, and numerous other genes to modulate DR-induced longevity, possibly through fat metabolism, autophagy, and other processes  mutants. One potential explanation for this observation is that by mutating nhr-62, the optimum DR threshold has been shifted to a new concentration. However, nhr-62(tm1818) mutants do not exhibit a globally shifted BDR response curve since the highest three food concentrations give median life spans that align precisely with wild-type. At further food dilution, nhr-62(tm1818) mutation significantly suppressed life span extension compared to wild-type, which results in a parallel response but with lower median life spans compared to wild-type. These results are consistent with a blunted response to DR in the lower nutrient range. In contrast to a “master regulator” of DR, which might completely abrogate life span extension at all food concentrations, this type of response curve could be indicative of a gene that plays a role in modulating or fine tuning the BDR longevity response at specific nutrient levels. It is also possible that nhr-62 mutation disrupts metabolism in a non-specific manner that is incompatible with a proper DR response. What argues against this is that nhr-62 mutation on its own had very little effect on gene expression or physiology under ad libitum conditions, suggesting nhr-62 is engaged in a regulatory role primarily under DR states.It was unexpected to see a partial BDR response by nhr-62 exhibits several phenotypes suggesting it functions in mobilizing fat to partition energetic and biosynthetic demands under nutrient limitation. Notably nhr-62 mutation prevents some of the reduction of TAG and Oil Red O staining stores seen under DR longevity. Several dynamic changes in fatty acid metabolism have been observed upon DR shifts in other species. In flies there is a shift toward increasing fatty acid synthesis and breakdown, and inhibiting this suppresses DR longevity nhr-62 alters fatty acid composition in the eat-2 background, increasing the amount of the saturated fatty acid palmitate, and reducing the amount of various MUFAs and PUFAs, including C18:1n7, C20:3n6, C20:4n6, C20:4n3, and C20:5n3 fatty acids. It is possible that these changes could contribute towards the suppression of life span. Interestingly, a handful of these fatty acids have been recently implicated in longevity. The omega-6 fatty acid, DGLA (C20:3n6) stimulates autophagy and extends C. elegans life span lipl-4, which stimulates autophagy C40H1.8 lipase could stimulate autophagy, since RNAi against this gene diminished DR longevity and GFP::LGG-1 punctual formation. Alternately, lipolysis could liberate fatty acids that serve a signaling role, binding to NHR-62 or related receptors. Future studies should further clarify the complex relationships between fatty acid composition, lipolysis, autophagy and longevity in the DR response.eat-2 to wild-type. Amongst enriched categories are genes involved in phosphorus metabolism, unsaturated fatty acid metabolism, eicosanoid and ceramide metabolism, lipid modification and transport, amino acid, amine and chitin metabolism, and neuropeptide signaling. Notably mutation of nhr-62 reversed the regulation of about 600 of these genes, suggesting a key role in mediating the DR response. These genes are candidates for nhr-62 targets, and possibly important effectors of the DR response , lipid binding , fatty acid remodeling (acyl coA thiolases F57F4.1), β-oxidation  and fatty acid elongation . Presumably some of these genes may mediate the physiologic changes relevant to DR. Indeed, vitellogenin knockdown has been previously shown to extend life By RNA-seq analysis we identified significant changes in the expression of approximately 3,000 genes comparing response . Among tlet-363/TOR RNAi induced longevity in the nhr-62 mutant background. We also found that the autophagy gene lgg-1 was up-regulated 1.5-fold, in a manner dependent on nhr-62. Consistently, we observed that autophagic vesicles visualized with GFP::LGG-1 were increased under DR in an nhr-62 dependent manner. Previous studies have shown that lgg-1 and other autophagy genes are required for DR and various longevity pathways Dietary restriction is associated with reduced TOR signaling, reduced protein synthesis, and increased autophagy inf-1, whose down-regulation has been shown to extend life span nhr-62 dependence. It is possible that these minor transcriptional changes collectively recapitulate the physiologic effect of DR. Alternately, modest regulation of a critical enzyme or key regulator in the pathway might be sufficient. nhr-62 might also regulate unidentified post-transcriptional modifiers of these processes, especially given the many kinases and phosphatases affected  and fungal induced proteins, which are speculated to be involved in innate immunity. In the future it will be important to understand how the diverse transcriptional output of nhr-62 relates to longevity and to identify direct targets by ChIP-seq.Reduced protein translation extends life span and may be one of the important outputs of the DR response affected ,S7. Manynhr-62. The C. elegans genome has undergone a radical expansion of the HNF4 family of nuclear receptors, making an assignment of homologous function for nhr-62 challenging. One such HNF4-like nematode receptor, nhr-49, has been proposed as PPARα-like because it is involved in the starvation response, turns on genes implicated in β-oxidation, fatty acid desaturation, binding and transport. However, in our hands, nhr-49 knockdown did not abrogate the DR response. Conceivably, nhr-62 functions similar to the PPARs as well, since these nuclear receptors are known lipid sensors that regulate metabolism under different nutritional states. Corton et al originally observed a substantial overlap in the expression profile of DR and PPAR nuclear receptor regulation in mice nhr-62 functions like HNF4, which regulates apolipoprotein levels and lipid and glucose homeostasis. Notably, both PPARs and HNF4 nuclear receptors can bind to fatty acids, which may be regulating their activity nhr-62 is involved in fat metabolism raises the possibility it too is regulated by fatty acid like ligands.It is currently unclear which mammalian homolog is most functionally analogous to C. elegans transcription factor SKN-1/NF-E2 regulates DR-induced longevity from the pair of ASI sensory neurons Interestingly, the E. coli (OP50) at 20°C unless otherwise noted. Standard procedures for culturing and maintaining strains were used eat-2(ad465), nhr-62(tm1818), nre-1(hd20);lin-15b(hd126), UL1385 mvEx5591(Y67A6A.2::gfp + unc-119), eat-2(ad465);mvEx5591(Y67A6A.2::gfp + unc-119), OP403 wgIs403(nhr-62::TY1 EGFP 3× FLAG (91H02);unc-119(+)); unc-119(tm4063), QU1 izEx1(plgg-1::gfp::lgg-1 + rol-6), eat-2(ad465); izEx1(plgg-1::gfp::lgg-1 + rol-6), eat-2(ad465);nhr-62(tm1818) dhEx627(pmyo-3::cfp + fosmid WRM065CF04). The dhEx627 extra-chromosomal rescue strain was generated by injecting DNA from worm fosmid WRM065CF04 (10 ng/µl), a co-injectable marker (pmyo-3::cfp at 20 ng/µl) and salmon sperm DNA at 70 ng/µl. Stable arrays were selected for and maintained based on expression of the co-injected marker. For imaging of nhr-62::gfp, day 1 adult worms were visualized using a Zeiss Axioskop2 Plus microscope and representative images were processed with ImageJ All strains were grown and maintained on NGM agar seeded with egg laying defective), in which unlaid eggs hatch inside the mothers, were censored from the aging experiment. The number of aging experiments performed, mean, median, and maximum lifespans, log-rank analysis, number of worms used in each experiment and worms censored were recorded (nre-1(hd20);lin-15b(hd126) was used. Eggs were pipetted at a target density of 15 eggs per well into 12 well cell culture plates with RNAi NGM seeded with RNAi clone of interest. At L4, or day 0 of adult, 15 µl of FUdR (5-Fluoro-2′-deoxyuridine) (50 mM) was pipetted into each well to inhibit progeny production. On day 7 of adulthood, the number of worms alive was determined for each well. On day 15 of adulthood, the number of worms alive was again determined and the percent surviving was calculated. For RNAi experiments, worms were maintained on NGM plates with 20 µg/ml carbenicillin, 10 µg/ml tetracycline, and 1 mM IPTG. Worms were transferred to fresh RNAi plates every other day. For BDR longevity experiments, lifespans were recorded as previously described 2HPO4, 6 g KH2PO4, 1 ml cholesterol at 5 mg/mL in ethanol, and MilliQ H2O to 1 L) with carbenicillin (50 µg/ml), kanamycin (10 µg/ml), and tetracycline (1 µg/ml) added to inhibit bacteria replication. 90 worms were then moved into defined bacteria concentrations and scored every 3–4 days for survival at a density of 15 worms per well. On every score day worms were transferred to freshly made bacteria condition. FUdR was added to the BDR liquid media for the first 14 days of adulthood to inhibit progeny production. Bacteria concentration was determined through serial dilution, plating, and counting of colony forming units (CFUs). The number of aging experiments performed, mean, median, and maximum lifespans, log-rank analysis, number of worms used in each experiment and worms censored were recorded  and the Pierce BCA Protein Assay Kit (Thermo Scientific) and analyzed on a Biotek Power Wave XS plate reader according to manufacturer's instructions. Three technical replicates were preformed for every biological replicate. Standards were also done in triplicate. Statistics were done using GraphPad Prism software.2SO4 (Sigma) in methanol (Biosolve) was added to each sample and incubated at 80°C for 20 minutes with shaking. 0.75 ml of water  and 350 µl of hexane (Sigma) were next added and the sample was incubated with shaking for 10 min to extract FAMEs into the hexane layer. The hexane layer was removed, concentrated by evaporation, and used directly for analysis. FAME analysis was carried out using an Agilent 7890A GC equipped with a flame ionization detector (FID)  and a DB-23 column  using helium as a carrier gas at a flow rate of 3 ml/min. 1 µl per sample was injected in pulsed-splitless mode. The initial oven temperature was set to 80°C, held for 1 minute; increased to 170°C at 6.5°C/min; then increased to 215°C at 2.75°C/min. FAMEs were identified and compared against known reference standards for quantification.Day 1 adults animals were collected in 100 µl M9 buffer, washed 2-times, and run through 10-freeze/thaw cycles before being sonicated to lyse the cuticle and tissue. Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific) according to manufacturer's instructions. 50 µl was removed to a 1.5 ml gas chromatography (GC) vial for derivatization. To each vial, 200 ng each of methyl C11:0, C13:0, and C23:0  were added as internal reference standards. To convert fatty acids into their fatty acid methyl ester (FAME) derivatives, 0.5 ml of 2.5% Hama-1 and cdc-42 was used as control. 4 biological replicates containing 300–400 worms each and four technical replicates were tested for each experiment. qPCR primers sequences are in Synchronized worms were collected into TRIzol (Invitrogen) when reaching adulthood but bearing no embryos. Total RNA and cDNA was prepared using RNeasy Mini kit (QIAGEN) and iScript cDNA Synthesis Kit (Bio-Rad) respectively. qRT-PCR was performed with Power SYBR Green master mix (Applied Biosystems) on a ViiA7 384 Real-Time PCR System (Applied Biosystems). A combination of nhr-62 RNAi experiments, 3 biological replicates with 31 animals (345 seam cells) of GFP::LGG-1 (L4440), 26 animals (225 seams cells) of GFP::LGG-1 (nhr-62i), 62 animals (773 seams cells) of eat-2 GFP::LGG-1 (L4440), and 58 animals (598 seam cells) of eat-2 GFP::LGG-1 (nhr-62i) were scored. For C40H1.8 RNAi experiments, 3 biological replicates with 50 animals (323 seam cells) of GFP::LGG-1 (L4440), 43 animals (382 seams cells) of GFP::LGG-1 (C40H1.8i), 100 animals (933 seams cells) of eat-2 GFP::LGG-1 (L4440), and 99 animals (896 seam cells) of eat-2 GFP::LGG-1 (C40H1.8i) were scored.Counting of puncta formation was done as previously described eat-2, nhr-62;eat-2, nhr-62, N2, giving a total of 12 samples. Sequencing reads were clipped to remove adapter sequence using fastx_clipper (http://hannonlab.cshl.edu/fastx_toolkit/), clipped reads were aligned to the C. elegans reference genome (WBcel215.67) using tophat version 1.3  with option −g1 to ensure unique mapping and option −G to pass the Caenorhabditis_elegans.WBcel215.67.gtf. The number of reads mapping to each Ensemble gene was counted with htseq-count (http://www-huber.embl.de/users/anders/HTSeq/doc/count.html#count). Statistical analysis and drawing of plots was performed in R (http://www.r-project.org/) using the bioconductor package Deseq  and R function gplots (http://cran.r-project.org/web/packages/gplots/index.html). Scatterplots of the RNA-seq data were drawn to illustrate differential expression between samples. A heatmap showing Euclidean distances between samples  was plotted in R. For each data comparison a list of “significant differentially expressed genes” was compiled from the Deseq-result as follows: FDR<0.05, mean normalized count >10 and absolute Foldchange (FC) >1.5. Furthermore a list of genes, which are up or down regulated by eat-2, compared to the control (eat-2 vs N2), but show that they are rescued in the nhr-62;eat-2 phenotype  was compiled and referred to as Rescued-Genes. Further analysis of “significant differentially expressed genes” and “Rescued-Genes” was performed as follows:Worms of indicated genotypes were synchronized by two rounds of bleaching and approximately 300 worms for each genotype were handpicked into TRIzol (Invitrogen) when they just reached adulthood without carrying embryos. Total RNA was prepared with RNeasy Mini Kit (QIAGEN). cDNA library was subsequently constructed by TruSeq RNA Sample Preparation Kit (Illumina). Illumina sequencing (100 bp single end) was performed on 3 biological replicates for each of the 4 treatments A Venn diagram was plotted in R for “significant differentially expressed genes” to show the overlap of the significant differentially expressed genes.Annotation and Gene-enrichment analysis of “significant differentially expressed genes” and “Rescued-Genes” was performed with David ReviGO analysis Figure S1nhr-49 by RNAi does not abolish longevity of eat-2(ad465) mutant animals (log-rank p<0.001). (B) Percentage of animals alive on day 15 fed bacteria expressing different RNAi constructs. nhr-49 RNAi resulted in shortevity. Knockdown of the nhr-62 paralogs nhr-21, nhr-4, nhr-34, and nhr-100 by RNAi does not suppress eat-2;nre-1;lin-15b longevity. (C) Median lifespan of wild-type (N2) or eat-2(ad465) worms fed nhr-62, nhr-4, or nhr-21 RNAi.Related NHRs do not mediate DR longevity response. (A) Knockdown of (TIF)Click here for additional data file.Figure S2nhr-62 overexpression. (A) Pumping rates of day 1 wild-type (N2) and wild-type(dhEx627) worms are not different. (B) Body length of day 1 adult wild-type(dhEx627) are shorter than wild-type (N2) worms. (C) Oil Red O staining of wild-type (N2) and wild-type(dhEx627). Oil Red O intensity in wild-type(dhEx627) is slightly decreased relative to wild-type (N2). ***p<0.001 by unpaired t-test. Mean (Center Line) ± SD (Box) with bars representing an outlier coefficient of 1.5.Physiologic traits of (TIF)Click here for additional data file.Figure S3nhr-62 mutation. (A) Pumping rates of day 1 adult eat-2(ad465) and eat-2;nhr-62 mutants are reduced relative to wild-type (N2) and nhr-62(tm1818) mutants (combined data from 2 independent experiments). (B) Relative body length of day 1 adult eat-2(ad465) and eat-2;nhr-62 mutants are smaller than ad libitum controls. (C) Day 1 adult eat-2(ad465) and eat-2;nhr-62 worms have reduced total progeny compared to ad libitum controls. nhr-62(tm1818) mutants have a modest but significant decrease in total hatched progeny compared to wild-type (N2) (combined data from 3 independent experiments). *p<0.05, **p<0.01, ***p<0.001 by Single Factor ANOVA with Tukey test. Mean (Center Line) ± SD (Box) with bars representing an outlier coefficient of 1.5.Physiologic traits of (TIF)Click here for additional data file.Figure S4fat-2, a Δ12-desaturase involved in PUFA synthesis. Levels of fat-2 mRNA were significantly up-regulated under DR as measured by qPCR. Mutation of nhr-62 partially suppressed this up-regulation, though this did not reach significance (p = n.s.). *p<0.05 by single factor ANOVA with Tukey test. Mean±SEM.Expression levels of (TIF)Click here for additional data file.Figure S5C40H1.8 partially suppresses DR-induced longevity.  C40H1.8 RNAi slightly reduces the lifespan of eat-2;nre-1;lin-15b worms relative to gfp RNAi fed controls (2 independent experiments). (C) Percent change in median lifespan of eat-2;nre-1;lin-15b relative to nre-1;lin-15b animals fed either C40H1.8 RNAi or gfp RNAi calculated from the experiments shown in A and B.(TIF)Click here for additional data file.Figure S6C40H1.8 and homologs. Level of (A) C40H1.7 mRNA (B) C40H1.8 mRNA and (C) C40H1.9 mRNA in wild-type (N2) worms fed C40H1.8 RNAi relative to wild-type (N2) worms fed gfp RNAi. *p<0.05 by student's unpaired t-test. Mean±SEM.qPCR of (TIF)Click here for additional data file.Figure S7C40H1.8. Sequence alignment of C40H1.8 to lipase class 3 proteins in various species. Catalytic triad (CT), nucleophilic elbow (NE), dashes represent breaks in the amino acid sequence. Accession numbers are C40H1.8:NP_001021213.1, Atg15p:EEU05398.1, Os11g0299300:ABA92846.1, C40H1.9:NP_001021214.2, diacyclglycerol lipase D. melanogaster:ACF37118.1, diacyclglycerol lipase M. musculus:NP_659164.2, and diacyclglycerol lipase H. sapiens:NP_631918.3.Sequence alignment of predicted lipase (TIF)Click here for additional data file.Figure S8Analysis of RNA-seq data. (A) Heatmap showing Euclidean distances between RNA-seq samples . (B) Venn diagram shows the number of distinct and overlapping regulated genes from RNA-seq. (C–N) qPCR validation of RNA-seq candidates. *p<0.05, **p<0.01, ***p<0.001 by single factor ANOVA with Tukey test. Mean±SEM.(TIF)Click here for additional data file.Table S1Plate Aging.(XLSX)Click here for additional data file.Table S2BDR Aging.(XLSX)Click here for additional data file.Table S3Genes tested in DR lipase screen.(XLSX)Click here for additional data file.Table S4eat-2 versus N2.Genes regulated in (XLSX)Click here for additional data file.Table S5eat-2 versus N2.Gene enrichment DAVID analysis of genes regulated in (XLSX)Click here for additional data file.Table S6nhr-62 rescued genes.(XLSX)Click here for additional data file.Table S7Gene enrichment DAVID analysis of rescued genes.(XLSX)Click here for additional data file.Table S8qPCR primers.(XLSX)Click here for additional data file.


We here revealed that hypo-responsiveness of CMV-specific late-stage CD8+ T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR) recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter.Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR) engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1 Adoptive T cell therapy recently showed significant success in the treatment of malignant diseases (CM) showed superior over effector memory T(EM) cells by proliferating more rapidly upon antigen encounter and by persisting longer upon adoptive transfer, thereby promoting repetitive migration into the draining lymph node and re-entering circulation + CD45RObright CD57+ CD7− late-stage phenotype With respect to the therapeutic efficacy central memory Tex vivo engineered with a transgenic TCR or alternatively with a chimeric antigen receptor (CAR) that in contrast to the TCR consists of a single polypeptide chain with an extracellular antibody-derived binding domain and the intracellular CD3-zeta signaling moiety To engraft adoptive T cell therapy with defined specificity, patient's T cells are We here elucidated that the hypo-responsiveness of the physiological TCR in terminally differentiated, late-stage T cells is due to membrane anchoring of TCR components whereas the down-stream signaling pathway is still functional. Consequently, late-stage T cells are susceptible to CAR CD3-zeta-mediated activation resulting in cytokine release and redirected cytotoxicity despite hypo-responsive TCR. Our data provide a rationale for CAR redirected T cell activation in the adoptive immunotherapy to avoid hypo-responsiveness upon repetitive antigen encounter.+ T cells from CMV infected, HIV−, HLA-A2+ individuals. CMV-specific T cells, identified by pp65 tetramer binding, predominantly consist of late-stage KLRG-1+ CD57+ CD7− T cells and a minor subset of intermediate-stage KLRG-1−/low CD57− CD7+ cells . Both T cell subsets, however, recruited CD2 into lipid rafts within the same time upon CD2/CD28 stimulation indicating that impaired TCR synapse formation in late-stage T cells is restricted to TCR components and not due to an overall decrease in membrane fluidity.We asked whether hypo-responsiveness of late-stage T cells is due to impaired TCR synapse formation. T cells were stimulated with the immobilized agonistic anti-CD3 antibody, alternatively with anti-CD2 antibodies, in presence of CD28 costimulation and TCR synapse formation was recorded by confocal microscopy. Within 5 min upon stimulation intermediate-stage T cells showed coalescence of TCR components into lipid rafts and TCR synapse formation in the contact region to antigen . Late-stWe asked whether impaired TCR synapse formation is due to reduced membrane mobility of TCR components as a consequence of galectin-3 binding to branched sugar residues as reported for other T cell subsets + cells and the cytolytic degranulation of cells indicated by CD107a staining  the TCR down-stream signaling pathway is functional and (ii) a CD3-zeta signaling CAR bypasses the TCR hypo-responsiveness redirecting late-stage T cells to a full anti-tumor cell response. We therefore engineered those T cells with a CAR with MHC-independent binding specificity for carcinoembryonic antigen (CEA) and the CD3-zeta signaling endodomain for T cell activation. Upon retroviral transduction T cells of the CD7ciencies . Life imciencies . Galecticiencies  No co-loytolysis . CAR-med+ T cells is due to down-regulated expression of TCR components, especially the CD3-gamma chain, following ligand triggering −/low CD57− CD7+ intermediate and KLRG-1+ CD57+ CD7− late-stage of terminal differentiation, express the TCR and bind specific tetramers with similar efficiencies; CD7− T cells, however, respond less efficiently with IFN-gamma and cytolytic degranulation than the CD7+ KLRG-1−/low CD57− T cells. While hypo-responsiveness of intermediate-stage T cells is due to blockade through PD-1/PD-1L interaction, this is not the case for late-stage T cells (unpublished data). Those T cells show impaired TCR synapse formation, which is due to a selective immobility of the TCR components in the cell membrane and likely mediated by galectin-3. Our conclusion is sustained by the observation that swainsonine, which prevents galectin-3 mediated anchoring of membrane proteins by preventing branched sugar formation + and CD8+ T cells, not classified further in these manuscripts according to the distinct stages of T cell differentiation Repetitive antigen engagement of adoptively transferred T cells will inevitably end in terminally differentiated late-stage T cells + T cells sufficient to induce clonal proliferation, acquisition of effector functions, and entry into the memory pool in the absence of additional stimuli A significant proportion of T cells from the peripheral blood of tumor patients and from tumor lesions belong to the terminally differentiated late-stage T cell subset Various physiological T cell subsets are impaired in TCR synapse formation including orally tolerized T cells While late-stage T cells can be rescued for use in the adoptive immunotherapy, their reduced proliferative capacity and their accelerated entry into apoptosis need to be addressed. In this regard CAR-redirected T cell therapy may be combined with recently developed strategies to sustain telomerase activity, which leads to improved cytotoxic capacities, cytokine and chemokine production. Alternatively, TNF-alpha production may be inhibited, which increases the proliferative potential and telomerase activity in those cells, or transgenic CD28 may be expressed, which retards replicative senescence + colon carcinoma cell line, and Colo320 (ATCC CCL 220.1) is a CEA− cell line. OKT3 (ATCC CRL 8001) is a hybridoma cell line that produces the anti-human CD3 monoclonal antibody (mAb) (OKT3); 15E8 hybridoma cells produce the anti-human CD28 mAb (15E8) . All cells were cultured in RPMI 1640 medium  containing 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 10% (v/v) heat-inactivated fetal calf serum (FCS) (Invitrogen).Buffy coats from healthy young volunteers (n = 30) (mean 30±10 years) were obtained from the blood bank facilities of the University Hospital Cologne, Germany. Volunteers who were taking immunosuppressive drugs or who had a disease potentially affecting the immune system were excluded. Peripheral blood from 8 patients (31–74 years) with confirmed diagnosis of active CMV and lack of HIV infection was analyzed. None of the CMV patients was on therapy at the time of sampling. All blood samples were taken after patients gave their written, informed consent. This study was performed in conformity with the Declaration of Helsinki of the World Medical Association and approved by the institutional review committee of the University of Cologne (reference no 02-041). 293T cells are human embryonic kidney cells that express the SV40 large T antigen (ATCC CRL 11268). LS174T (ATCC CCL 188) is a CEAThe following antibodies were used: APC-conjugated anti-TCR-alpha/beta(IP26) , PE-Cy7-conjugated anti-CD8 mAb (RPA-T8) , APC-conjugated anti-CD45RO mAb (UCHL-1) (BD Biosciences), FITC-conjugated anti-CD7 mAb (4H9) (BD Biosciences), APC-conjugated anti-CD7 mAb (MEM-186) , APC-Cy7-conjugated anti-CD45RO mAb (UCHL-1) (BD Biosciences), APC-Cy7-conjugated anti-CD3 mAb (UCHT-1) (BD Biosciences), Alexa-488-conjugated anti-KLRG-1 mAb (13A2) , APC-Cy7-conjugated anti-CD8 mAb (SK1) (BD Biosciences), APC-conjugated anti-CD57 mAb (TB03) , PE-Alexa-750-conjugated anti-CD7 mAb (CD7-6B7) , and PE-Cy7-conjugated anti-CD45RO mAb (UCHL-1) (BD Biosciences). To monitor intracellular galectin-3 expression T cells were stained with PE-conjugated anti-CD7 mAb (4H9) (BD Biosciences), fixed and permeabilized, and subsequentially stained with FITC-conjugated anti-galectin-3 mAb  . Appropriate isotype antibodies (BD Biosciences) were used as controls. 15E8 and OKT3 mAbs were affinity purified from hybridoma supernatants utilizing goat anti-mouse IgG2a antibodies  immobilized on N-hydroxy-succinimid-ester-(NHS)-activated sepharose . Cells were analyzed by flow cytometry using a FACS Calibur or FACSCanto cytometer (BD Biosciences), equipped with Cell Quest or FACS DIVA software (BD Biosciences). Viable lymphocytes were gated according to forward/side scatter and lack of 7-AAD (BD Biosciences) staining. Human recombinant interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) were purchased from R&D Systems . IFN-gamma in the culture medium was recorded by ELISA utilizing matched pairs of specific antibodies . The detection limit of the assay is 15 pg/ml IFN-gamma.+ CD45RO+ T cells were obtained from peripheral blood mononuclear cells by negative depletion magnetic cell sorting (MACS) (Miltenyi Biotec) using the “CD8+ T cell isolation kit” and anti-human CD45RA microbeads. CD8+ CD45RO+ T cells were separated into CD7+ and CD7− subpopulations by using the FITC-conjugated anti-human CD7 antibody (4H9) (BD Biosciences) and anti-FITC microbeads (Miltenyi Biotec). CD8+ T cells were obtained from peripheral blood mononuclear cells by positive selection using anti-human CD8+ microbeads (Miltenyi Biotec). The purity of the isolated T cell subpopulations was routinely >95%.CD85 cells) into 293T cells for virus production. T cells were activated by addition of IL-2  and OKT3 mAb (100 ng/ml) for 48 hrs and incubated with retroviruses for additional 48 hrs. CAR expression was monitored by flow cytometry using a PE-conjugated F(ab′)2 anti-human IgG1 antibody (1 µg/ml) (Southern Biotechnology), which recognizes the extracellular IgG1 CH2CH3 domain of the CAR, and a FITC-conjugated anti-human CD3 mAb (UCHT-1) (BD Biosciences). After 24 hrs without stimuli redirected cytolysis was recorded by incubating T cells in increasing numbers with tumor cells (2.5×104 per well) for 48 hrs in round-bottom 96-well plates, specific cytotoxicity monitored by a XTT-based colorimetric assay  and the viability of tumor cells was calculated as follows:The generation of the retroviral expression cassettes for the CEA-specific CAR BW431/26scFv-Fc-CD3-zeta(#439) and retroviral transduction of T cells was described in detail + T cells from CMV infected, HIV− donors were stained for HLA-A2 with mAb (BB7.2) (BD Biosciences). Tetramer-binding T cells were detected by incubation with the PE-conjugated MHC tetrameric complexes HLA-A*0201/pp65495–503 . Intracellular IFN-gamma was detected in PBMC's from CMV patients stimulated for 6 hrs in presence with PE-conjugated MHC tetrameric complexes HLA-A*0201/pp65495–503 (Sanquin) together with CMV peptide NLVPMVATV (“CMV pp65 Peptide Mix”) or the TCR cross-linking reagent “Cytostim” (both from Miltenyi Biotec) or HIV peptide SLYNTVATL , respectively, and stained for CD8, CD7 and CD45RO. During the final 5 hrs, brefeldin A (5 µg/ml) (BD Biosciences) was added to avoid release of cytokines from the Golgi apparatus, respectively, and cells were subsequently stained for IFN-gamma using the clone 25723.11 mAb (BD Biosciences). Alternatively, lymphocytes were activated for 6 hrs in presence of FITC-conjugated anti-CD107a mAb (H4A3) (BD Biosciences), the CMV peptide, and the agonistic anti-CD49d mAb (L25) (1 µg/ml) plus the anti-CD28 mAb (L293) (1 µg/ml) (both from BD Biosciences).CD85 cells) were placed on cover slips coated with poly-L-lysine (0.1 mg/ml) , the anti-CD3 mAb (UCHT-1) and the anti-CD28 mAb (CD28.2) or with the anti-CD2 mAbs (L303.1) and (L304.1) and the anti-CD28 mAb (CD28.2), or as control by an isotype-matched IgG1 . Alternatively, cover slips were coated with the BW2064/36 mAb (10 µg/ml), which is an internal image anti-idiotypic antibody, directed against the anti-CEA scFv BW431/26 T cells (10Results are presented as means ± standard error of the mean (SEM). A paired and unpaired two-tailed t-test was used; p<.05 was considered significant.Figure S1CMV-specific T cells in late-stage and intermediate-stage of terminal differentiation equally bind pp65 CMV tetramers. CMV-specific CD8+ T cells in late or intermediate stage of terminal differentiation were identified in the peripheral blood from CMV patients with acute virus reactivation by incubation with PE-conjugated CMV peptide loaded tetramers HLA-A*0201/pp65495–503 and staining for CD8, CD7, CD45RO, CD57, KLRG-1, CD3 and TCR-alpha/beta as described in (PDF)Click here for additional data file.Figure S2− late-stage T cells are hypo-responsive to TCR/CD3 stimulation.CD7 The CD7− and CD7+ subsets of CD8+ CD45RO+ T cells were isolated from the peripheral blood and cultured (105 cells/100 µl) with or without the agonistic anti-CD3 mAb (OKT3) (5 µg/ml), the anti-CD28 mAb (15E8) (5 µg/ml), or with IL-2 (50 U/ml). (A) IFN-gamma in the culture supernatant at day 6 was detected by ELISA. (B) To monitor CD107a+ IFN-gamma producing cells, cells were monitored by flow cytometry by staining with the PE-conjugated anti-IFN-gamma mAb (25723.11) and the FITC-conjugated anti-CD107a mAb (H4A3). Assays (− cells compared with the corresponding CD7+ T cells.. Assays  & B were(PDF)Click here for additional data file.


HTLV indeterminate WB patterns are frequently observed in plasma/serum samples from persons living in intertropical areas.In the framework of ongoing projets on HTLV-1/2 and related viruses in central Africa, we systematically analysed by WB, plasma from villagers living in south Cameroun. The studied group included 2155 individuals , either Bantous (1258) or Pygmies (897). All plasma samples were tested by WB (HTLV 2-4 MPD) with interpretation done according to manufacturer instructions. Only clear bands were considered as positive/informative. DNA extracted from buffy-coat were subjected to PCR using several primer pairs known to detect HTLV-1/2/3/4. Positive PCR bands were sequenced.Among the 2155 plasma samples, 48 were HTLV-1, 20 HTLV-2, and 134 HTLV. Furthermore, 955 were indeterminate including 100 HGIP (HTLV-I Gag-indeterminate pattern) [Search for the origin of this frequent new WB is ongoing with special insights concerning cross-reactivities with parasitic antigens as suggested for the HGIP pattern .


It resulted in a dramatic increase of CD11b+Gr1-low frequency and a significant decrease of the frequency of CD11b+Gr1-high and CD4-CD8-NK1.1+ cells in the recipient’s spleen. Unexpectedly, donor IL-6 deficiency could not significantly reduce macrophage frequency irrespective of in the host’s spleen or graft. Taken together, suppression of innate immune effector cells and enhanced activity of regulatory MDSCs contributed to tolerance induction by blockade of IL-6 signaling pathway. The unveiled novel mechanism of targeting IL-6 might shed light on clinical therapeutic application in preventing accelerated allograft rejection for those pre-sensitized transplant recipients.Engraftment of IL-6 deficient donor into wild-type recipient could significantly improve allograft survival through T cell lineage particularly regulatory T cells (Tregs) in non-sensitized transplant host. However, its effect on innate immune responses remains uncertain. Our data revealed that donor IL-6 deficiency significantly increased infiltration of two subsets of MDSCs (CD11b+Gr1+myeloid-derived suppressor cells), CD11b+Gr1 Interleukin-6 (IL-6) as a pleiotropic cytokine can be produced by various tissues particularly proinflammatory cell types and formed in local cytokine milieu. It is capable of orchestrating cellular differentiation, affecting early graft function recovery , and dis+CD25+ regulatory T cells (Tregs), and an initiation of T-cell immunoresponses against graft [Indeed, not only adaptive but innate immune cells are required for acute or chronic rejection in various transplant settings . A recenst graft . By contAlthough impact of IL-6 on adaptive immune responses was described in different transplant models ,3,7, itsb; Stock No. 000664), BALB/c , and B6:129S2-IL-6tm1Kopf/J . The mice were housed under pathogen-free conditions at the Center for Life Science . Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at Beth Israel Deaconess Medical Center. Allogeneic heterotopic heart transplantations (HTx) were performed using BALB/c donors and C57BL/6 recipients.Eight-twelve week male inbred mice were obtained from the Jackson Laboratories  including wild-type C57BL/6 . All mAbs used for cell surface staining were purchased from BioLegend . For FACS staining, anti–mouse CD4  FITC-conjugated mAb, anti–mouse CD8a  PE-conjugated mAb, Alexa Fluor® 700-conjugated anti-mouse Gr-1/Ly-6G  mAb, and the pacific blue-conjugated anti–mouse CD11b  mAb were used to label live T cells. All samples were acquired using an LSRII . Data was analyzed using FlowJo 7.5 software , as we reported before . The anaThe heart grafts were harvested, minced and then incubated in Roswell Park Memorial Institute (RPMI) 1640 medium containing 0.5 mg/mL collagenase IV  for 30 min at 37°C in the shaker. Tissue debris was carefully removed with 70-µm cell strainer. After washing twice, viable mononuclear cells were separated by Ficoll density gradient centrifugation. The cells at the interface were isolated and washed thrice with RPMI 1640 medium supplemented with 10% FBS and then subjected to either fluorescence-activated cell sorting (FACS) staining.Cardiac grafts were harvested at day 9 post-transplantation. Heart specimens were fixed in 10% paraformaldehyde and then embedded in paraffin. Tissue was sectioned at 5µm-thickness. These sections were stained with hematoxylin and eosin (H&E) and assessed by light microscopy to determine the extent of cellular infiltration.Frozen heart graft specimen were sectioned at 5-μm-thickness and immunohistochemistry was performed using an avidin-biotin-peroxidase technique , with ImmPACT DAB Chromogen. Heart sections were blocked with goat serum and then labeled with an anti- Mouse Ly-6G , a monoclonal antibody , which recognizes neutrophils. Specimen sections were incubated using the Avidin-Biotin Complex Solution, Vectastain Elite ABC kit . ImmPACT DAB Chromogen was applied and subsequently counter-stained with Harris hematoxylin. Allograft survival curves were generated by using the Kaplan-Meier method. All data were analyzed using the statistical software GraphPad Prism version 5.0 . The FACS data between groups were determined by a student T-test. The difference of graft survival between different groups was compared by both log-rank (Mantel-Cox Test) and Gehan-Breslow-Wilcoxon Test. A p-value of .05 was determined as statistical significance. The FACS data were expressed as mean ± SD.Previously, various experimental studies demonstrated a critical role of donor IL-6, which mainly originated from the donor allograft tissue and could impair allograft survival . This daHowever, additional use of anti-CD25 mAb (PC61) to those IL-6-/- heart grafts (IL-6-/-BALB/c) pronouncedly shortened graft survival equivalent to allogeneic control group (B6 BALB/c) . An incr-low, CD11b+Gr1-int, and CD11b+Gr1-high with distinct immunoregulatory effect. Therefore, we tried to characterize MDSCs subsets, macrophage, NK1.1+ cells in the spleen of wild-type donor + PCI (G5) and IL-6 deficient donor + PCI (G6) by flow cytometry analysis (-low frequency (p=0.0021) and a significant decrease of CD11b+Gr1-high (p=0.00015) and CD4-CD8-NK1.1+ cells (p=0.0017) frequency in the recipient’s spleen , which have been thoroughly studied in our previous research. MDSCs can be sub-grouped into three different subsets, CD11b+Gr1analysis . As a res spleen . -low and CD11b+Gr1-int with strong immunosuppression activities, the pre-sensitized transplanted grafts with PCI including IL-6-deficiency and wild-type were harvested. The infiltrated leukocytes within graft were isolated and subject to cytometry analysis. The findings revealed that donor IL-6 deficiency did not remarkably suppress infiltration of macrophage within graft (p=0.3), while it caused a significant increase of intragraft CD11b+Gr1-low (p=0.00001) and CD11b+Gr1-int frequencies (p=0.032) in the early stage . Thereafter, it is reasonable to explain that remarkable infiltration of two main subsets of MDSCs, CD11b+Gr1-low and CD11b+Gr1-int was observed within donor IL-6 deficient graft. A dramatic increase of CD11b+Gr1-low frequency and a significant decrease of CD11b+Gr1-high frequency were found in the recipient’s spleen. Therefore, our present study tested the role of those innate immune cells including macrophage, NK1.1+ cell, and MDSCs subsets in the setting of PCI-mediated injury in the pre-sensitized transplant host. The findings manifested that donor IL-6 deficiency mounted a weaker immune response in such pre-sensitized transplant recipient. At day 3 post-transplantation macrophage frequency in the graft  and spleBy far, it is well-studied that heart is the main source producing cytokine IL-6 . The infTaken together, our present study revealed that blockade of IL-6 signaling pathway suppressed innate immune effector cells in large measure and enhanced activity of regulatory MDSCs. This unveiled novel mechanism of targeting IL-6 might shed light on clinical therapeutic application in preventing accelerated allograft rejection for those pre-sensitized transplant recipients.


Biomarkers offer the possibility to speed up diagnosis of ventilator-associated pneumonia (VAP) and differentiate it from nonpulmonary infection. One such marker, the triggering receptor expressed on myeloid cells-1 (TREM-1), exists as a soluble protein and a surface receptor expressed on monocytes and neutrophils [Paired bronchoalveolar lavage (BAL) and blood were obtained from 25 VAP patients, 15 ventilated non-infected controls, 10 ventilated patients with nonpulmonary infection and 25 nonventilated controls. VAP diagnosis was by clinical pulmonary infection score (CPIS) and semiquantitative microbiology. BAL and blood monocytic and neutrophilic levels of surface TREM-1 and CD11b (leukocyte activation marker) were assessed using flow cytometry. Monocytes were CD14-positive. Soluble TREM-1, IL-1β, IL-6 and IL-8 were measured using ELISA. BAL dilution was corrected by urea assay.See Figure The BAL/blood ratio of monocytic surface TREM-1 diagnoses VAP and differentiates pulmonary from nonpulmonary infection. CD14 and TREM-1 may have a role in the pathogenesis of VAP.


Arabidopsis, the phytohormone gibberellin (GA) stimulates stamen development by opposing the DELLA repression of B and C classes of floral homeotic genes. GA can promote male flower formation in cucumber (Cucumis sativus L.), a typical monoecious vegetable with unisexual flowers, and the molecular mechanism remains unknown. Here we characterized a DELLA homolog CsGAIP in cucumber, and we found that CsGAIP is highly expressed in stem and male flower buds. In situ hybridization showed that CsGAIP is greatly enriched in the stamen primordia, especially during the hermaphrodite stage of flower development. Further, CsGAIP protein is located in nucleus. CsGAIP can partially rescue the plant height, stamen development and fertility phenotypes of Arabidopsis rga-24/gai-t6 mutant, and ectopic expression of CsGAIP in wide-type Arabidopsis results in reduced number of stamens and decreased transcription of B class floral homeotic genes APETALA3 (AP3) and PISTILLATA (PI). Our data suggest that monoecious CsGAIP may inhibit staminate development through transcriptional repression of B class floral homeotic genes in Arabidopsis.In hermaphroditic  Hordeum vulgare), rice (Oryza sativa), Mirabilis jalapa and Pharbitis, implying that GA may be required for flower opening Arabidopsis, rice and tomato (Solanum lycopersicum), whereas stimulate pistillate development in castor bean (Ricinus communis), Hyoscyamus and maize (Zea mays) Gibberellins (GAs) are one class of tetracyclic diterpenoid phytohormones that play essential roles in diverse aspects of plant growth and development, including seed germination, hypocotyl elongation, root growth, stem elongation, leaf expansion, trichome formation, floral induction, flower development, and fruit development CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO) and ent-kaurenoic acid oxidase (KAO) GIBBERELLIN-INSENSITIVE DWARF1 (GID1) genes  in Arabidopsisgid1 display no or partial GA-deficient phenotypes respectively, triple mutant showed severe GA-deficient abnormality, including extremely dwarfism, delayed flowering, incomplete floral organs and GA-insensitivity GID1 results in early flowering GID1 promotes the interaction between GID1 and DELLA proteins, which leads to rapid degradation of DELLA proteins through the SLY1/GID2SCF  ubiquitin-proteasome pathway, and the proteolysis of DELLA proteins releases their inhibitory effect on GA-responsive genes and allows plant growth and development Arabidopsis: RGA (REPRESSOR OF ga1-3), GAI (GIBBERELLIN INSENSITIVE), RGL1 (RGA-LIKE 1), RGL2 (RGA-LIKE 2), and RGL3 (RGA-LIKE 3) RGA and GAI are negative regulators for stem elongation RGA and RGL2 coordinately inhibit the development of petal, stamen and anther, while RGL1 exacerbates this repression APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG), while removing the RGL2 and RGA DELLA activities in ga1-3 mutant (ga1-3 rgl2-1 rga-t2) can rescue the phenotypes of flower development, including delayed flowering time, aberrant petal, stamen and anther development, suggesting that GA regulates flower development via degradation of DELLA proteins, especially RGA and RGL2, thus allows the transcription of floral homeotic genes GAMYB, on the other hand, acts as a positive regulator for GA signaling pathway GAMYB in Arabidopsis (myb33myb65) results in shorter filaments, pollen abortion and male sterile, similar to the GA-insensitive phenotype GAMYB is involved in programmed cell death (PCD) of tapetal cells, exine and ubisch body formation, as well as in the GA-induced anther development Several key enzymes have been identified to be involved in GA biosynthesis, such as copalyl diphosphate synthase (Cucumis sativus L.) is a typical monoecious vegetable with individual male and female flowers, and has been served as a model system for sex determination in plantaGID1, DELLA and GAMYB have much higher expression than those of GA synthesis genes during male flower development, and the cucumber DELLA homolog CsGAIP has the highest expression. We cloned the CsGAIP and characterized its spatial and temporal expression patterns. CsGAIP is mainly expressed in stems and male flower buds, and CsGAIP protein is located in nucleus. Ectopic expression of CsGAIP can partially rescue the phenotypes of rga-24/gai-t6 double mutant in Arabidopsis, and overexpression of CsGAIP in wild type resulted in reduced number of stamens and decreased transcription of B class floral homeotic genes. Our results suggested that CsGAIP inhibits stamen development through transcriptional repression of B class floral homeotic genes in Arabidopsis.However, so far, most GA-regulated flower development studies were performed in hermaphroditic species, and rarely in monoecious plants. Cucumber  and GAMYB have much higher expression than those of GA synthesis genes CPS, KS, KO, KAO during cucumber male flower development. In which, GAIP has the highest expression among all, particularly in the hermaphrodite stage (stage 5), for example, expression of GAIP is more than 20 fold and 6 fold higher than GA synthesis genes and other GA signal transduction factors, respectively. Further, expression of GAIP decreases as the male flower develop, suggesting that cucumber GAIP may play a key role during male flower development and promote male determination in the hermaphrodite stage.The developmental process of cucumber male flower can be divided into 12 stages DELLA homologs in cucumber, CsGAIP (Csa021618), CsGAI1 (Csa015919), CsGAI2 (Csa008181) and CsGAI3 (Csa015258), in which CsGAIP has the highest similarity to DELLAs in Arabidopsis, so we chose CsGAIP for further analysis in this study. CsGAIP was cloned using cDNA derived from cucumber leaves through PCR technology. The full-length CsGAIP cDNA consists of 1761 bp and encodes 587 amino acids. Consistent with the five DELLA genes of Arabidopsis, CsGAIP gene also has no introns Arabidopsis, cucumber, pumpkin (Cucurbita maxima), lettuce (Lactuca sativa), pea (Pisum sativum), bean (Phaseolus vulgaris), and monocotyledon (red line) such as maize, rice, barley and wheat. Within dicotyledon clade, CsGAIP and CmGAIP, which belong to the cucurbitaceae family with unisexual flowers, fall into the same clade that is distinct from those of CsGAI2, CsGAI3 and other DELLA homologs in hermaphroditic species, such as Arabidopsis, lettuce, pea and bean, implying that CsGAIP may be involved in the unisexual flower development in cucumber.Through BLAST analysis, we found four DELLA homologs transcripts, qRT-PCR was performed in various cucumber tissues including roots, stems, leaves, male flower buds, female flower buds and fruits. As shown in CsGAIP and CsGAI2 are much higher than those of CsGAI1 and CsGAI3 in all the tissues we examined, and that CsGAIP and CsGAI2 display similar expression patterns, which are predominantly expressed in stems and male flower buds. CsGAI3 transcript is more enriched in roots as compared to other tissues, while CsGAI1 shows equivalent expression in all the tissues we tested. Among all the four DELLA homologs, CsGAIP displays the highest expression especially in stems and male flower buds, implicating that CsGAIP may play important roles in stem and male flower development.To characterize the spatial distribution of CsGAIP during male flower development of cucumber by in situ hybridization (CsGAIP RNA was found throughout in the inflorescence meristem (im) and floral meristem (fm)  , as welltem (fm)  in stageorocytes , anther orocytes , despiteorocytes . As negastage 12 .Arabidopsis, the DELLA proteins RGA and GAI have been shown to contain putative nuclear localization signal (NLS) and localize in nucleus Allium cepa) (In um cepa) , top rowCsGAIP, we ectopically expressed the full-length CsGAIP cDNA under the control of 35S promoter of Cauliflower mosaic virus (CaMV) in Arabidopsis rga-24/gai-t6 double mutant, and 13 independent transgenic lines were obtained. Previous study reported that rga-24/gai-t6 double mutant displayed higher plant height, reduced number of pollens, shorter filaments and thus decreased seed numbers per silique rga-24/gai-t6 phenotypes. The average plant height of rga-24/gai-t6 plants is 38% taller than that of Ler, while in the transgenic lines, the average plant height is only 8% taller than that of Ler  , APETALA2 (AP2), APETALA3 (AP3), PISTILLATA (PI) and AGAMOUS (AG), are involved in floral patterning in ArabidopsisAP3 and PI) and C gene (AG) are down-regulated by RGA activity 35S::CsGAIP plants by qRT-PCR and semi-quantitative RT-PCR. We found that the expression of A class (AP1 and AP2) and C class of gene (AG) were not substantially changed in the transgenic plants, but transcripts of B function genes (AP3 and PI) were significantly decreased  is a monoecious species with individual male and female flowers. During the early stage of flower development, both stamen primordia and carpel primordia are initiated, male or female flower is generated upon the arrestment of carpel or stamen development, respectively Arabidopsis, GA promotes stamen development by antagonizing the function of DELLA proteins DELLA homolog may play important roles during male flower development in cucumber , CsGAI1 (Csa015919), CsGAI2 (Csa008181) and CsGAI3 (Csa015258), with CsGAIP, CsGAI2 and CsGAI3 closely relate to RGA and GAI, and CsGAI1 likely to be the homolog for RGL1-3  development. Genetic transformation in cucumber upon CsGAIP RNA interference or overexpression would shed light on the molecular function of CsGAIP during sex determination of cucumber flowers. Meanwhile, F (CsACS1G) and M (CsACS2) genes have been demonstrated to regulate unisexual flower development in cucumber, specifically, F gene promotes female flower development M gene inhibits stamen development in floral buds CsGAIP, F and M during sex determination in future studies. In addition, in monoecious maize, GA causes feminization instead of staminate production In cucumber, exogenous GA treatment can promote male flower formation rimordia , 3 and 4RGA and GAI in Arabidopsis results in higher plant height and earlier flowering DELLA homologs REDUCED HEIGHT and DWARF8 leads to dwarfism in wheat and maize, respectively DELLA homologs have conserved role in stem elongation, but the specific role maybe even opposite in different species. In this study, CsGAIP is highly expressed in cucumber stems , suggesting that monoecious CsGAIP has divergent functions from RGA and GAI in hermaphroditic Arabidopsis.Loss of function of er stems  and that4/gai-t6 , Table 24/gai-t6 , Table 2n in Ler , Table 3f CsGAIP , similarArabidopsis DELLAs have specific as well as partially overlapping roles, it would be interesting to explore the specificity of the function for each DELLA homologue in cucumber. The four cucumber DELLAs display distinct expression patterns , SCL3 (SCARECROW-LIKE 3), ALC (ALCATRAZ) and JAZs (JA ZIM-domain proteins) Given that patterns , in whicCucumis sativus L.) line 3461 was used in this study. The plants were grown in a growth chamber under 16 h/8 h and 25°C/18°C in day/night, respectively. Upon two true-leaf stage, plants were transferred to a greenhouse in the experimental field of China Agricultural University in Beijing. The Arabidopsis mutant rga-24/gai-t6 (Landsberg background) was provided by Sun's lab Ler was used as wild type control. Arabidopsis seeds were germinated on Murashige-Skoog (MS) medium, which contains 1% sucrose and 0.2% phytagar at 4°C for 3 days and then moved to 22°C under a regime of 16 h light/8 h dark. Seedlings were transferred to soil 7–10 d after germination.A monoecious cucumber (5′-ATGAAGAGGGAGCATCACCATCTTC-3′) and CsGAIP-R (5′-TCACTTAGCGACCACCGGGTT-3′) at 95°C for 5 min; 30 cycles of 95°C for 30 s, 54°C for 30 s, and 72°C for 2.5 min; and then 72°C for 10 min. The amino acid sequence of related DELLA proteins were obtained from National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) or the Arabidopsis Information Resource (http://www.arabidopsis.org), and protein alignment of CsGAIP and related DELLAs was performed using ClustalW in the MEGA5 software package, and the boxes were drawn using the BoxShade web site (http://www.ch.embnet.org/software/BOX_form.html). The phylogenetic tree was constructed using the neighbor-joining (NJ) method Total RNA was extracted from cucumber leaves using the Promega's SV Total RNA Isolation System, and cDNA was synthesized using MultiScribe reverse transcriptase (Applied Biosystems). The cDNA was amplified with primers CsGAIP-F (Premix Ex Taq from TaKaRa (China) on an Applied Biosystems 7500 real-time PCR system. The cucumber α-tubulin gene (TUA) and Arabidopsis β-tubulin gene (TUB2) were used as internal references. For semi-quantitative RT-PCR, the β-tubulin gene (TUB2) was used as a control. Both qRT-PCR and semi-quantitative PCR were repeated in three independent samples. The gene primers for qRT-PCR were as follows: CPS-F (5′-GCTGAGGTCAATGGACGATG-3′) and CPS-R (5′-TGAGAATATTTGACTGTCACCCC-3′); KS-F (5′-CAATGGTCCCTTCTCCAAACT-3′) and KS-R (5′-CCCATCGCTTAAGAGTAAGAACAC-3′); KO-F (5′-AAGAGGCTAT- GGTGACGAGGTA-3′) and KO-R (5′-ACATGAGCAAACAACTCCCTAGA-3′); KAO-F (5′-CACTCAAGGCTCGGAAGAATC-3′) and KAO-R (5′-CAACATCAATCAGAGCGTCCAT-3′); GID1-F (5′-ATCCAGCATGTAATCCCTTCG-3′) and GID1-R (5′-CCATCATTCTCCAGCCCTCT-3′); CsGAIP-F (5′-GCTCAAACGCATTCAAACAAG-3′) and CsGAIP-R (5′-GCTATGAGTGGGCGAGTGTG-3′); CsGAI1-F (5′-GCCGTCCACTACAACCCTTCC-3′) and CsGAI1-R (5′-GTCCACGAGACACTCCCATCC-3′); CsGAI2-F (5′-TAAAGACGACGAAGCCGAAGATA-3′) and CsGAI2-R (5′-AATAAACCTCCGACAACAACACG-3′); CsGAI3-F (5′-GGAGGAAGACCACGACAAGCATC-3′) and CsGAI3-R (5′-CGGAGTATTGAGTTCAGCGAGCA-3′); GAMYB-F (5′-TCTAACCCTACCACAAAGAACGC-3′) and GAMYB-R (5′-TCTATCTGGTGCCAACACAAAAGT-3′); TUA-F (5′-ACGCTGTTGGTGGTGGTAC-3′) and TUA-R (5′-GAGAGGGGTAAACAGTGAATC-3′); AP1-F (5′-GTTGCTCTTGTTGTCTTCTCCC-3′) and AP1-R (5′-CTCCATCGACCAGTTTGTATTG-3′); AP2-F (5′-GGTGTTGCTTCTGGCTTTCCT-3′) and AP2-R (5′-GTCCACGCCGACTCTTTTTCA-3′); AP3-F (5′-TATTTCTGATGTCGATGTTTGGGC-3′) and AP3-R (5′-ACTTTTGTTCTTTTTCTTGGTGGT-3′); PI-F (5′-TGGATTGGTGAAGAAGGCTAA-3′) and PI-R (5′-GATCTCCATCTGGTGGTCTCG-3′); AG-F (5′-ATAATCAGCATACAAAACTCCAAC-3′) and AG-R (5′-ATACTTCTCTCTAATCTGCCTTCC-3′); TUB2-F (5′-ATCCGTGAAGAGTACCCAGAT-3′) and TUB2-R (5′-AAGAACCATGCACTCATCAGC-3′). The primers of AP1, AP2, AP3, PI and AG for semi-quantitative PCR were performed as previously reported Total RNA was extracted using Promega's SV Total RNA Isolation System, and cDNA was synthesized using MultiScribe reverse transcriptase (Applied Biosystems). Quantitative real-time RT-PCR (qRT-PCR) was performed using SYBR CsGAIP in situ probes were as follow: GATTTAGGTGACACTATAGAATGCTATCCGATGCCTAATTTTGCGA-3′5′- (bold represents the SP6 RNA polymerase binding sites) and TGTAATACGACTCACTATAGGGGCATCTGAAGCCTATCGGACACT-3′5′- (bold shows the T7 RNA polymerase binding sites).Shoot apex of 10-day-old seedling and male flower buds from 45-day-old cucumbers grown in the greenhouse were fixed and hybridized as described onion epidermal cells, the full length coding region of CsGAIP were cloned using primers GTCGACATGAAGAGGGAGCATCACCATCTTC-3′5′-ACGC  and GGATCCCTTAGCGACCACCGGGTTGTT-3′5′-CG (BamH I site in bold), and then inserted into the pEZS-NL vector (with GFP protein driven by 35S promoter) to generate 35S:GFP-CsGAIP, and the empty pEZS-NL vector was used a control. The constructs were introduced into cucumber protoplasts using Huang's method For transient expression in cucumber protoplasts and CsGAIP overexpression construct, full length CsGAIP cDNA were cloned using primers ACTAGTATGAAGAGGGAGCATCACCATCTTC-3′5′-GG (Spe I site in bold) and CACG TGTCACTTAGCGACCACCGGGTT-3′5′-GACTGC (Pml I site in bold), and inserted into the pCAMBIA1305.1 vector with 35S promoter. The construct was then introduced into Agrobacterium by electroporation and transformed into Ler or rga-24/gai-t6 plants as described To make the CsGAIP (Csa021618), CsGAI1 (Csa015919), CsGAI2 (Csa008181), CsGAI3 (Csa015258), AtRGA (At2g01570), AtGAI (At1g14920), AtRGL1 (At1g66350), AtRGL2 (At3g03450), AtRGL3 (At5g17490), CmGAIP (Q6EI06), ZmD8 (Q9ST48), TaRHT1 (Q9ST59), HvSLN1(Q8W127), OsSLR1(Q7G7J6), LsDELLA1 (BAG71200), LsDELLA2 (BAG71201), PsLA (ABI30654), and PvGAI2 (BAF62637).Sequence data in this study can be found in the Cucumber Genome DataBase, Arabidopsis Genome Initiative or GenBank/EMBL/Swiss-Prot databases under the following accession numbers:


The possibility that vaccination with Adenoviral vectors increased mucosal T-cell activation remains a central hypothesis to explain the potential enhancement of HIV acquisition within the STEP trial. Modeling this within rhesus macaques is complicated because human Adenoviruses, including Adenovirus type 5 (HAd5), do not productively infect macaques. We created a vector based upon a naturally occurring rhesus macaque Adenovirus (SAdV7) to test whether vaccination with a species-specific Adenoviral vector enhances mucosal T-cell activation within the natural host.Twelve rhesus macaques were vaccinated 3x intramuscularly with SAdV7 vector. Five HAd5-vaccinated animals were included as controls. PBMC and rectal biopsies were obtained at baseline, multiple times post-prime and post-17 week boost , and post-31 week second boost . We assessed rectal mucosal lamina propria and blood for frequency changes of Ad-specific T-cell responses and T-cell activation levels by measuring IFNg, TNFa, IL2, CD25, Ki67, CD69, and HLA-DR.Naturally acquired pre-existing SAdV7-specific CD4+ T-cells were identified in 10/13 macaques within blood and/or rectal mucosa. Following intramuscular SAdV7 vaccination, rectal SAdV7-specific CD4+ T-cell responses increased above baseline in 9/9 animals 2-5 weeks post-prime, and subsequently contracted. Five weeks post-prime, 10/12 animals had rectal SAdV7-specific CD4+ T-cell responses ranging from 0.1-16.84%. As expected, SAdV7-specific CD4+ T-cells expressed CD69 and other activation markers (but not Ki67). Heightened expression of CD25, CD69, and HLA-DR was observed on total rectal memory CD4+ T-cells in SAdV7-vaccinated animals, and maintained 15 weeks after the prime. Interestingly, upregulation of activation markers in rectal mucosa also occurred in HAd5-vaccinated animals. No change in activation was observed in the blood throughout the entire study.These results indicate that peripheral vaccination with an Adenovirus vector can increase the activation of mucosal CD4+ T-cells providing an experimental model to further evaluate the role of host-vector interactions on increased HIV acquisition.


During plant embryogenesis, once the suspensor organ of the plant embryo has fulfilled its role, it is removed by programmed cell death (PCD). The pro-death cathepsin protease NtCP14 initiates this PCD, but is inhibited by the cystatin NtCYS until the suspensor function is fulfilled. Nicotiana tabacum) embryogenesis as a model system to demonstrate that the mechanism triggering suspensor PCD is based on the antagonistic action of two proteins: a protease inhibitor, cystatin NtCYS, and its target, cathepsin H-like protease NtCP14. NtCYS is expressed in the basal cell of the proembryo, where encoded cystatin binds to and inhibits NtCP14, thereby preventing precocious onset of PCD. The anti-cell death effect of NtCYS is transcriptionally regulated and is repressed at the 32-celled embryo stage, leading to increased NtCP14 activity and initiation of PCD. Silencing of NtCYS or overexpression of NtCP14 induces precocious cell death in the basal cell lineage causing embryonic arrest and seed abortion. Conversely, overexpression of NtCYS or silencing of NtCP14 leads to profound delay of suspensor PCD. Our results demonstrate that NtCYS-mediated inhibition of NtCP14 protease acts as a bipartite molecular module to control initiation of PCD in the basal cell lineage of plant embryos.Plant zygote divides asymmetrically into an apical cell that develops into the embryo proper and a basal cell that generates the suspensor, a vital organ functioning as a conduit of nutrients and growth factors to the embryo proper. After the suspensor has fulfilled its function, it is removed by programmed cell death (PCD) at the late stages of embryogenesis. The molecular trigger of this PCD is unknown. Here we use tobacco . This is especially apparent during plant embryogenesis, when a plant zygote divides into an apical cell, which develops into a mature embryo, and a basal cell, which generates a single organ called the suspensor. During early seed development, the suspensor connects the embryo to the surrounding seed tissues and transports the nutrients and hormones required for its early development. Once its functions are fulfilled, the suspensor is subsequently eliminated by PCD. In this study, we answer a long-standing question in the field by elucidating the mechanism that is responsible for initiating suspensor PCD at a specific time during embryogenesis. Our findings show that in tobacco plant embryos, suspensor PCD is controlled by two antagonistically acting proteins—the pro-death cathepsin protease NtCP14 and its inhibitor cystatin NtCYS, which co-localize to the basal-most cell of the suspensor. High expression levels of  Arabidopsis embryos, respectively, molecular regulation of suspensor PCD remain elusive. In particular, molecular trigger responsible for the timely initiation of cell death in the suspensor is unknown.Plant development begins with asymmetric division of the zygote, giving rise to two daughter cells with distinct developmental fates. A small apical cell is the founder of cell lineage generating the embryo proper, whereas a larger, basal cell establishes cell lineage leading to the embryo-suspensor. The function of the suspensor is to connect embryo to the surrounding seed tissues and to transport nutrients and growth factors to the embryo proper N. tabacum) embryos provide a tractable model system to address the molecular mechanisms of suspensor PCD, because of the formation of a single file of four or five suspensor cells through a highly stereotyped and precisely timed sequence of cell divisions, beginning from the first zygotic division  because of its strictly basal cell-specific expression pattern. Rapid amplification of cDNA ends (RACE) followed by three rounds of genome walking produced a 683-bp cDNA and a 4,247-bp upstream region containing an ATG initiation codon. Analysis of the ORF indicated that the gene contains a complete ORF of 420 nucleotides encoding a protein of 140 amino acids. BLASTP search returned several matches to proteins containing a cystatin-like domain and a cystatin-specific QxVxG motif, indicating that the gene is a new member of the cystatin family in N. tabacum  and human cathepsin L (Ki = 0.39±0.04 nM), no measurable inhibition of human cathepsin B was detected. These data establish NtCYS as a member of the cystatin family with inhibitory activity against a subset of cysteine proteases.Most cystatins can inhibit activity of cysteine proteases from the papain C1A family NtCYS, RNA was extracted from sperm cells, egg cells, zygotes, two-celled proembryos, and eight-celled, 32-celled, and heart embryos, and NtCYS expression was determined using semi-quantitative reverse transcription PCR (RT-PCR). NtCYS transcripts were detected in all samples, except for sperm cells and heart embryos . NtCYS was highly expressed in two- and four-celled proembryos  and in the eight-celled embryos (stage 3), whereas the expression was barely detectable at more advanced stages of embryogenesis and was absent in non-embryonic organs  and was barely detectable in embryos at stage 4 and 5 . The nuclear envelope appeared intact until globular-to-heart transition (stage 7), when the nucleus became lobed and crenulated . These eRecent evidence suggests the involvement of proteases with caspase 1, 3, and 6-like activities in various types of PCD in plants NtCYS expression in the embryos (NtCYS using three independent RNA interference (RNAi) constructs expressed under the native promoter and selected six homozygous RNAi lines for detailed analysis . Precocious activation of PCD in the basal domain caused abortion of 20.7% to 40% of seeds in different NtCYS-deficient lines . Furthermore, this anti-cell death role of NtCYS is crucial for the formation of a functional suspensor and completion of embryogenesis.NtCYS encodes a cystatin, which inhibits papain and human cathepsin L in vitro, we investigated whether cathepsin-like activity correlates with NtCYS expression in tobacco proembryos. For this, we compared proteolytic activities of the total protein extracts prepared from two-celled proembryos in WT and NtCYS RNAi plants using the fluorescent peptides Z-FR-AMC (for cathepsin L-like proteases), Z-RR-AMC (for cathepsin B-like proteases), and Bz-FVR-AMC (for cathepsin H-like proteases). Silencing of NtCYS led to 1.4- and 2.6-fold increase in proteolytic activity towards Z-RR-AMC and Bz-FVR-AMC, respectively, but did not affect activity towards Z-FR-AMC . Next, we used bimolecular fluorescence complementation (BiFC) analysis in the transiently co-transfected tobacco epidermal cells to study interaction between NtCYS and the cysteine proteases, which revealed interaction with six proteases, named NtCP3, NtCP6, NtCP8, NtCP14, NtCP15 , and NtC3RX3FX2NX3I/VX3N)  , which i3I/VX3N)  and S3.NtCYS RNAi plants  = 0.74±0.18 µM)  .Allium cepa) epidermal cells, and found that both proteins are cytoplasmic . In contrast to WT, a large proportion (77% to 81% for different lines) of 32-celled (stage 4) embryos in the NtCYS-overexpressing lines had TUNEL-negative suspensors  do not have aspartate cleavage specificity NtCYS in tobacco embryos poses an intriguing question regarding how the proteolytic activity of NtCP14 is controlled in the other suspensor cells, as well as in the cells of the apical cell lineage, which are destined to survive but devoid of NtCYS . Total RNA was isolated from seeds at 5 d after pollination and used as a template to synthesize first-strand cDNA with the SMART RACE cDNA Amplification Kit (Clontech). The NtCYS and NtCP14 promoters were isolated using the Genome Walker DNA walking method with the Genome Walker Universal Kit (Clontech). All reactions were performed according to the manufacturer's instructions.A full-length cDNA for A multiple sequence alignment of the known cystatin family genes in dicot plants was conducted using CLUSTALX v. 1.81 with the default multiple alignment parameters. The phylogenetic tree was constructed with PHYLIP v. 3.68.mRNA isolation from sperm cells, egg cells, zygotes, and embryos, and cDNA synthesis were performed as described previously NtCYS lacking signal peptide sequence was inserted into an NdeI/XhoI -digested pMXB-10 vector (NEB). The resulting plasmid was transformed into Escherichia coli BL21 (DE3). The recombinant NtCYS protein was expressed and purified according to the manufacturer's instructions. The purified NtCYS was re-purified by anion exchange chromatography with a Bio-Scale Mini UNOsphere Q Cartridge (Bio-Rad) using a linear gradient of NaCl (0–300 mM) in Tris-HCl (20 mM [pH 8.0]) on BioLogic DuoFlow system (Bio-Rad). Expression and purification of cysteine proteases were performed according to The coding region of i values for the interaction of NtCYS with cysteine proteases, papain (Sigma-Aldrich), human liver cathepsin L (Sigma-Aldrich), and human liver cathepsin B (Sigma-Aldrich) substrate hydrolysis progress curves were monitored. Papain activity was measured in 50 mM phosphate buffer (pH 6.0), using the synthetic fluorogenic substrate Z-FR-AMC (Sigma-Aldrich). Human cathepsin L activity was measured in 400 mM sodium acetate buffer (pH 5.5), using the synthetic fluorogenic substrate Z-FR-AMC. Cathepsin B activity was measured in 50 mM phosphate buffer (pH 6.0), using the substrate Z-RR-AMC (Sigma-Aldrich). Hydrolysis proceeded at 25°C (cathepsin L) or 37°C (papain and cathepsin B) with 40 µM substrate after or without the addition of NtCYS under reducing conditions. The activity levels were monitored using a Spectra Max M2 (Molecular Device Co.) with excitation and emission filters of 360 and 455 nm, respectively.For determination of the Ki(app) of NtCYS for each recombinant cysteine protease was assessed according to the following equation: Ki(app) = [I0]/(V0/Vi−1).To determine the optimal substrates for the recombinant cysteine proteases, the proteases were activated in a buffer containing 100 mM sodium acetate (pH 4.0), and their activities were measured using substrates Z-FR-AMC (Sigma-Aldrich), Z-RR-AMC (Sigma-Aldrich), Bz-FVR-AMC (Bachem), L-R-AMC (Sigma-Aldrich), Z-AAN-AMC (Bachem), Ac-YVAD-AMC (Sigma-Aldrich), Ac-DEVD-AMC (Sigma-Aldrich), and Ac-VEID-AMC (Bachem). Hydrolysis proceeded at 30°C with 40 µM substrate in each reaction (pH 6.0) under reducing conditions. The KTo perform endogenous protease activity assays, the embryos were frozen in 15 mM sodium phosphate or sodium acetate buffer with 0.1% Brij-35 at −80°C until use in protease assays. Proteolytic activity was measured in 10-µl reaction mixtures containing the proteins released from unfrozen embryos, and 125 µM of individual substrates, 1 mM EDTA, 10 mM L-Cys, and 0.01% Brij-35 in 15 mM sodium phosphate or sodium acetate at 30°C. The amount of AMC released was determined by capillary electrophoresis. Detailed procedure for capillary electrophoresis is described in Standard methods for analyzing cell viability and DNA fragmentation are also described in supplemental information In situ detection of caspase-like activity in the embryos was performed using Carboxy fluorescein FLICA Apoptosis Detection kits , which include cell-permeable fluorochrome-conjugated inhibitors of caspases. Detailed procedure is described in HindIII and EcoRI were ligated into the plant transformation vector pCAMBIA1300, resulting in pCAMBIA-SPYNE and pCAMBIA-SPYCE. All full-length ORFs (without stop codons) of cysteine protease genes were inserted in-frame into the vector pCAMBIA-SPYCE. Similarly, the NtCYS gene was inserted into the vector pCAMBIA-SPYNE.BiFC constructs were prepared using the vectors pSPYNE-35S and pSPYCE-35S Agrobacterium tumefaciens strain GV3101, which was used to infiltrate N. benthamiana leaves. The transient expression was assayed according to Sparkes All BiFC constructs were transferred into N. benthamiana by Agrobacterium-mediated transient expression. GFP was used as the negative control. Infiltrated leaves of N. benthamiana were collected and ground into powder. Extracts for co-IP were prepared at 4°C in a buffer containing 15 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, and protease inhibitor cocktail . Next, a Chromotek GFP-trap (Allele Biotech) was used to capture the GFP-tagged proteins according to the manufacturer's instructions. The immunoprecipitates were then subjected to SDS-PAGE and the target protein was transferred to nitrocellulose membranes and probed with anti-NtCYS  and anti-GFP . NtCYS polyclonal rabbit antiserum was produced against recombinant NtCYS (GenScript). Co-IP experiment was repeated independently three times.NtCYS and NtCP14-GFP were co-expressed in A. cepa) epidermal cells through particle-mediated transient transformation using PDS-1000/He instrument (Bio-Rad). For subcellular localization, NtCYS-GFP and NtCP14-GFP were co-expressed with ER marker containing an N-terminal signal peptide derived from an Arabidopsis vacuolar basic chitinase and the C-terminal amino acid sequence HDEL (RFP-ER) For co-localization analysis, NtCYS-GFP and NtCP14-RFP were co-expressed in onion . Images were processed with Image J or Adobe Photoshop.4 (0.25%) in 30% ethanol overnight and embedded in Spurr's resin. Ultrathin sections were post-stained with uranyl acetate/lead citrate and examined with a transmission electron microscope (FEI Tecnai G2 20 TWIN).For ultrastructural examination, tobacco seeds were fixed with glutaraldehyde (2.5%) in PBS buffer (100 mM [pH 7.4]) for 24 h, dehydrated in a graded ethanol series, post-fixed with OsOhttp://www.ncbi.nlm.nih.gov/Genbank) under following accession numbers: NtCYS (KF113570), NtCP3 (Z99173), NtCP6 (KF113571), NtCP8 (KF113572), NtCP14 (KF113573), NtCP15 (KF113574), and NtCP23 (AB032168).Sequence data used in this work can be found in the GenBank  Alignment of NtCYS with closely-related members of cystatin family, including G. max GmCYS-6 (accession number ACU14962), M. truncatula MtCYS-1 (ABD28732.1) and MtCYS-3 (ABD32914.1), and P. trichocarpa PtCYS-7 (XP_002308795), PtCYS-8 (XP_002307893), and PtCYS-11 (EEF07232). Identical amino acid residues are boxed. Cystatin-like domain is labelled with a shaded box. Black dots indicate QxVxG motif. (B) A bootstrap consensus of phylogenetic tree representing similarities of NtCYS protein sequence with those of Arachis hypogaea AhCYS (accession number: AAU21498), A. thaliana AtCYS-1 (AT5G12140), AtCYS-2 (AT2G31980), AtCYS-3 (AT3G12490), AtCYS-4 (AT4G16500), AtCYS-5 (AT5G47550), AtCYS-6 (At3g12490), AtCYS-7 (At5g05110), B. campestris BcCYS (S65071), B. oleracea BoCYS-1 (ABD64998), BoCYS-2 (ABD64972), BoCYS-3 (ABD64929), BoCYS-4 (AAL59842), Castanea sativa CsCYS (CAA11899), G. max GmCYS-1 (ACU14306), GmCYS-2 (CAI84599), GmCYS-3 (CAI84598), GmCYS-4 (BAA19610), GmCYS-5 (ACU19522), GmCYS-6 (ACU14962), GmCYS-7 (CAI84604), GmCYS-8 (CAI84601), Helianthus annuus HaCYS-1 (JE0308), HaCYS-2 (BAA95416), Ipomoea batatas IbCYS (AAD13812), Malus x domestica MdCYS-1 (AAO18638) and MdCYS-2 (AAO19652), M. truncatula MtCYS-1 (ABD28732.1), MtCYS-2 (ABD28593.2), MtCYS-3 (ABD32914.1), M. sativa MsCYS (AAZ98791.1), Pyrus communis PcCYS (AAB71505.1), P. trichocarpa PtCYS-1 (EEF09526), PtCYS-2 (EEE98761), PtCYS-3 (EEE82959), PtCYS-4 (XP_002336760), PtCYS-5 (XP_002319462), PtCYS-6 (XP_002314231), PtCYS-7 (XP_002308795), PtCYS-8 (XP_002307893), PtCYS-9 (XP_002303955), PtCYS-10 (XP_002301749), PtCYS-11 (EEF07232), PtCYS-12 (ABK94227), Ricinus communis RcCYS-1 (EEF36180), RcCYS-2 (XP_002525552), RcCYS-3 (XP_002523225), RcCYS-4 (EEF31214), Solanum lycopersicum SlCYS-1 (AAF23126), SlCYS-2 (ABG23376), SlCYS-3 (AAF23127), SlCYS-4 (ABY83981), SlCYS-5 (AAF23128), S. tuberosum StCYS-1 (AAA16120), StCYS-2 (ABA40456), Vigna unguiculata VuCYS-1 (AAQ14319), VuCYS-2 (CAA79954), Vitis vinifera VvCYS-1 (XP_002283400), VvCYS-2 (XP_002271940), VvCYS-3 (XP_002277752), VvCYS-4 (XP_002274494), VvCYS-5 (XP_002267841), VvCYS-6 (XP_002268378), VvCYS-7 (XP_002265736), VvCYS-8 (CAN70300), VvCYS-9 (CAN62864). The tree was calculated with Phylip Ver. 3.68. software using Protpars method. The A. thaliana AtCYS-6 sequence was used as an outgroup sequence in order to root the phylogenetic tree. The subclan of NtCYS is labeled with shaded box.(TIF)Click here for additional data file.Figure S2Sequence alignment of tobacco cathepsins. Identical amino acid residues are boxed. Black dots indicate non-contiguous ERFNIN motif.(TIFF)Click here for additional data file.Figure S3Purification and substrate specificity of tobacco cathepsins. (A) Coomassie blue stained SDS-PAGE gel showing purified cathepsins. (B) Proteolytic activities of recombinant cathepsins against different substrates. The activity of each cathepsin against different substrates is expressed as the percentage of its activity against substrate FR-AMC. Data represent the mean ± SE of three independent experiments.(TIF)Click here for additional data file.Figure S4Co-localization analysis of NtCYS-GFP and NtCP14-GFP with Golgi and ER markers in onion epidermal cells. While no apparent co-localization was observed with Golgi marker ST-RFP (A), both proteins showed strong co-localization with ER marker RFP-ER (B). Scale bars, 50 µm.(TIF)Click here for additional data file.Figure S5Expression pattern of NtCP14. (A) Semi-quantitative RT-PCR analysis of NtCP14 in sperm cell (1), egg cell (2), zygote (3), and two-celled proembryo (4). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. (B) RT-qPCR analysis of NtCP14 in the embryos at stages 1 to 9, and in both floral and vegetative tissues. The expression level of NtCP14 in the embryos at stage 1 was set to 1. (C) Promoter activity of NtCP14 (proNtCP14::H2B-GFP expression) during embryogenesis. Scale bars, 10 µm.(TIF)Click here for additional data file.Figure S6Overexpression of cathepsin L-like genes driven by promoter proZC1. Enhanced expression of cathepsin L-like genes in transgenic lines, as measured by RT-qPCR. The expression level in the WT was set to l, except for NtCP8. Data represent the mean ± SE from three independent experiments. ** indicates statistical difference compared to WT .(TIF)Click here for additional data file.Figure S7Overexpression of NtCP14 driven by proNtCYS induces precocious cell death in the basal cell lineage. (A–C) Cell viability and nuclear DNA fragmentation at developmental stages 1 (A), 2 (B), and 3 (C) from WT and NtCP14-overexpressing line L1-4. Scale bars, 10 µm.  The frequency of the proembryos (stages 1 and 2) and eight-celled embryos (stage 3) with PI-positive (D) and TUNEL-positive (E) basal cells in WT and NtCP14-overexpressing line L1-4. Data represent the mean ± SE from three independent experiments, with 30 proembryos or embryos per line analyzed in each experiment (n = 90). ** indicates statistical difference compared to WT .(TIF)Click here for additional data file.Figure S8Upregulation of NtCYS or downregulation of NtCP14 delays the onset of suspensor PCD. (A) The frequency of suspensors containing indicated numbers of TUNEL-positive nuclei in NtCYS-overexpressing line L-2 at stages 4 to 8. Data represent the mean ± SE from three independent experiments, with 30 embryos per stage analyzed in each experiment (n = 90). (B) The frequency of suspensors containing indicated numbers of TUNEL-positive nuclei in NtCP14 RNAi line L3-15 at stages 4 to 8. Data represent the mean ± SE from three independent experiments, with 30 embryos per stage analyzed in each experiment (n = 90). (C–G) Representative examples of TUNEL-stained embryos in the NtCP14 RNAi lines at stages 4 to 8, respectively. Scale bars, 20 µm.(TIF)Click here for additional data file.Figure S9NtCYS-GFP expression delays the onset of suspensor PCD. (A) Expression of NtCYS-GFP driven by proZC1 in two-celled proembryos. Scale bars, 10 µm. (B) The frequency of suspensors containing indicated numbers of TUNEL-positive nuclei in NtCYS-GFP expressing line L-1 at stages 4 to 6. Data represent the mean ± SE from three independent experiments, with 30 embryos per stage analyzed in each experiment (n = 90). (C) The frequency of suspensors containing indicated numbers of TUNEL-positive nuclei in NtCYS-GFP expressing line L-2 at stages 4 to 6. Data represent the mean ± SE from three independent experiments, with 30 embryos per stage analyzed in each experiment (n = 90). (D–F) Representative examples of TUNEL-stained embryos in the NtCYS-GFP expressing lines at stages 4, 5, and 6, respectively. Scale bars, 10 µm. Asterisks indicate the basal cell.(TIF)Click here for additional data file.Figure S10Expression of NtCP14-GFP induces precocious cell death. (A) The mRNA level of NtCP14 in pNtCP14::NtCP14-GFP transgenic lines. The expression level of NtCP14 in WT was set to l. (B) The frequencies of PI-positive two-celled proembryos in WT and NtCP14-GFP expression lines. Data represent the mean ± SE from three independent experiments with 50 proembryos per line analyzed in each experiment (n = 150). (C) PI-FDA stained two-celled proembryos from WT and pNtCP14::NtCP14-GFP line. Scale bars, 10 µm.(TIF)Click here for additional data file.Text S1Additional details for methods.(DOC)Click here for additional data file.


Myocardial T1 mapping remains a challenging task due to restrictions imposed by cardiac and respiratory motion. Modified Look-Locker Inversion Recovery (MOLLI) [Figure SAPPHIRE T1-mapping was compared to MOLLI in phantom measurements and in healthy volunteers. A bottle phantom with a T1 of ~1300 ms was imaged using both T1-mapping sequences at various simulated ECGs with different heart-rates. Furthermore, pre-contrast T1-maps in five healthy volunteers were acquired using SAPPHIRE T1-mapping and MOLLI.In the phantom measurements SAPPHIRE T1-mapping is in good agreement with MOLLI measurements at a simulated heart-rate of 60 bpm (Relative difference: <2%). The SAPPHIRE T1-times, as depicted in Figure SAPPHIRE T1-mapping enables heart rate independent myocardial T1-mapping. The heart-rate invariance is achieved by applying a combination of saturation and inversion pulses as magnetization preparation.Deutsche Telekom Stiftung; NIH:R01EB008743-01A2; NIH: K99HL111410-01