Caenorhabditis elegans, slow fat consumption has been suggested to contribute to the extension of the survival rate during nutritionally adverse conditions. Here, we investigated the potential role of pyruvate dehydrogenase kinase (PDHK)-2, the C. elegans homolog of mammalian PDK, effects on fat metabolism under nutritional conditions. PDHK-2 was expressed at low levels under well-fed conditions but was highly induced during long-term starvation and in the dauer state. This increase in pdhk-2 expression was regulated by both DAF-16 and NHR-49. Dauer-specific induction of PDHK-2 was abolished upon entry into the post-dauer stage. Interestingly, in the long-term dauer state, stored fat levels were higher in daf-2(e1370);pdhk-2 double mutants than in daf-2(e1370), suggesting a positive relationship between PDHK-2 activity and fat consumption. PDHK-2 deficiency has been shown to lead to greater preservation of residual fats, which would be predicted to contribute to survival during the dauer state. A test of this prediction showed that the survival rates of daf-2(e1370);pdhk-2(tm3075) and daf-2(e1370);pdhk-2(tm3086) double mutants were higher than that of daf-2(e1370), suggesting that loss of either the ATP-binding domain (tm3075) or branched chain keto-acid dehydrogenase kinase domain (tm3086) of PDHK-2 leads to reduced fat consumption and thus favors increased dauer survival. This attenuated fat consumption in the long-term dauer state of C. elegans daf-2 (e1370);pdhk-2 mutants was associated with concomitant down-regulation of the lipases ATGL (adipose triglyceride lipase), HSL (hormone-sensitive lipase), and C07E3.9 (phospholipase). In contrast, PDHK-2 overexpression in wild-type starved worms induced lipase expression and promoted abnormal dauer formation. Thus, we propose that PDHK-2 serves as a molecular bridge, connecting fat metabolism and survival under nutritionally adverse conditions in C. elegans.In  Caenorhabditis elegansC. elegans, in particular, the critical role of insulin/insulin-like growth factor-1 (IGF-1) signaling (IIS) in metabolism and the control of aging has been well established. For example, suppression of the IIS pathway has been shown to cause a substantial increase in both mean lifespan (MLS) and fat accumulation C. elegans encounters unfavorable growth conditions  or high concentrations of dauer pheromones (daumones or ascarosides), it can enter the dauer state C. elegans have suggested that this process is compartmentalized according to metabolic phase and growth time C. elegans to survive an extended dormant period It has been well documented that a close relationship exists between energy metabolism and aging processes in animals, including the nematode The dauer state is resistant to environmental stresses, such as heat and cold, as well as oxidative stress, and the metabolic activity of dauer larvae is generally reduced C. elegans homolog of mammalian PDK-4 C. elegans under the nutritionally adverse conditions. To this end, we used two different C. elegans pdhk-2 mutants—pdhk-2(tm3075), which lacks the C-terminal ATP-binding domain, and pdhk-2(tm3086), which lacks the N-terminal mitochondrial branched chain α-ketoacid dehydrogenase (BCDH) kinase domain  compared to the wild-type N2 strain (20.5±1.8 days)  and daf-7(e1372) mutant worms using total mRNA prepared from L4-stage worms grown under well-fed conditions. We found that the expression level of pdhk-2 did not significantly differ in daf-2(e1370) and daf-7(e1372) mutant worms compared to N2 worms (pdhk-2 expression levels were unchanged in nhr-49(nr2041) mutants, which lack a nuclear hormone receptor involved in fatty acid metabolism, and daf-16(mu86) mutants, which lack the IIS downstream transcriptional regulator, DAF-16  . Second,d tm3086 , C. BecaN2 worms . Similar, DAF-16 .pdhk-2 expression in N2 worms grown under starvation conditions. We found a substantial increase (4.2-fold) in pdhk-2 gene expression in 12-hour starved groups compared to well-fed N2 worms (pdhk-2 expression was increased by 8-fold in the dauer state (induced by 7 days starvation of N2 on NGM plates at 20°C) compared to controls  and daf-7(e1372) mutants also showed enhanced pdhk-2 expression  upon entry into the complete dauer state . This induction of pdhk-2 expression in starvation and in the dauer state was not observed in either of the pdhk-2 mutants, tm3075 and tm3086 (data not shown).Because the expression of mammalian PDK-4 is known to increase during fasting N2 worms . Furthercontrols . This daC. elegans homolog of DAF-16, induces PDK-4 expression in mammalian skeletal muscle during starvation C. elegans NHR-49 C. elegans, we measured pdhk-2 mRNA levels in these mutants and N2 control worms that had been maintained under 12-hour starvation conditions. In contrast to the well-fed state, where mutant worms showed no significant changes in pdhk-2 expression compared to N2 controls (pdhk-2 were more than 2.0-fold lower in daf-16(mu86) and nhr-49(nr2041) mutants than in N2 controls (1.7 and 2.1 for daf-16(mu86) and nhr-49(nr2041), respectively, vs. 4.2 for N2; pdhk-2 expression under long-term starvation conditions. To examine the epistatic relationship between DAF-16 and NHR-49 with respect to PDHK-2 activation, we measured the expression levels of daf-16 and nhr-49 mRNA in N2 controls and daf-16(mu86) and nhr-49(nr2041) mutant strains under 12-hour starvation conditions. Whereas the expression of nhr-49 in daf-16(mu86) mutant was reduced compared to N2 with 12-hour starvation  mutant were not significantly changed (p = 0.1695). These results suggest that DAF-16 epistatically regulates NHR-49, while both DAF-16 and NHR-49 induce PDHK-2 under starvation conditions.It has been previously reported that the mammalian F-box protein, FOXO1, the controls , with lopdhk-2 promoter::GFP (pdhk-2p::GFP) and pdhk-2p::PDHK-2::GFP (PDHK-2::GFP). We found that pdhk-2p::GFP  primarily in the form of TGs, which are usually hydrolyzed by TG lipases C. elegans dauer larvae during the long-term dauer state heavily depends on the utilization of stored fat, the metabolic regulation of fats  is a key factor in determining survival rate pdhk-2 expression in the dauer state, we investigated the potential role of PDHK-2 in the survival of worms in the dauer state using daf-2(e1370);pdhk-2(tm3075) and daf-2(e1370);pdhk-2(tm3086) double mutants. Our goal was to correlate changes in fat content, if any, with changes in the MLS of these mutants under well-fed conditions and in short-term (day 1) and long-term (day 10) dauer states.Animals store fats in adipose tissue (intestines in pdhk-2 single mutants (daf-2(e1370);pdhk-2(tm3075) and daf-2(e1370);pdhk-2(tm3086) double mutants  and daf-2(e1370) control worms (45.5±1.3 days) (daf-2(e1370);pdhk-2 double mutants and daf-2(e1370) worms (data not shown).First, in the well-fed state, as was previously observed in the  mutants , there w.3 days) . Oil Red.3 days) . Moreovedaf-2(e1370);pdhk-2 double mutants and daf-2(e1370) control worms measured by Oil Red O staining (fats) and biochemical assays (TG levels) (daf-2(e1370);pdhk-2(tm3075) and daf-2(e1370);pdhk-2(tm3086) double mutants compared to daf-2(e1370) worms  , indicat0) worms , suggestdaf-2(e1370) controls and daf-2(e1370);pdhk-2(tm3075) and daf-2(e1370);pdhk-2(tm3086) double mutants revealed more residual TGs in the bodies of daf-2(e1370);pdhk-2 double mutants  compared to daf-2(e1370) worms (54.5%). These results imply that fat consumption is significantly attenuated by a deficiency of PDHK-2 activity.Third, measurements of fats remaining in the body of daf-2(e1370);pdhk-2 double mutants influenced the survival rate of dauer larvae. We found that the survival rate, measured as 50% survival population (S50), was higher for daf-2(e1370);pdhk-2(tm3075) , daf-2(e1370);pdhk-2(tm3086)  double mutants and daf-7(e1372) (18.8±0.89 days p<0.0001) compared to daf-2(e1370) controls ;pdhk-2(tm3075) and daf-2(e1370);pdhk-2(tm3086) double mutants at different dauer-state durations. In both short-term and long-term dauer groups, total lipase activity was substantially reduced in the double mutants (28–43%) compared to daf-2(e1370) controls (daf-2(e1370) (data not shown). Loss of either the ATP-binding (tm3075) or BCDH (tm3086) domain of PDHK-2 in daf-2;pdhk-2 double mutants appeared to similarly contribute to this concomitant down-regulation of PDHK-2 and lipase activity (daf-2(e1370) long-term (day 10) dauers (daf-2(e1370);pdhk-2(tm3075) and daf-2(e1370);pdhk-2(tm3086) double mutants during the long-term dauer state, indicating that these lipases may respond specifically to the suppression of PDHK-2 activity  dauers . Notablyactivity .C. elegans, we microinjected pdhk-2p::PDHK-2::GFP into N2 worms and the pdhk-2 mutants, tm3075 and tm3086; N2 injected with pdhk-2p::GFP was used as a control. After growing under well-fed or starved conditions, we examined MLS and dauer formation rate. Well-fed worms showed no significant difference in MLS following PDHK-2 overexpression: N2 control (pdhk-2p::GFP), 16.4±2.1 days; N2;PDHK-2/OE, 17.7±2.0 days; tm3075;PDHK-2/OE, 18.5±1.5 days; and tm3086;PDHK-2/OE, 18.4±2.0 days (daf-2(e1370) worms overexpressing PDHK-2  exhibited a reduction in the rate of dauer formation compared to daf-2(e1370) control worms (daf-2(e1370);pdhk-2p::GFP). That is, most daf-2(e1370) worms at the L1 stage typically entered dauer formation within 3 days (>60 hours) at 25°C, whereas transgenic daf-2(e1370);PDHK-2/OE worms displayed slow growth, abnormal dauer formation, and even bypassed the dauer stage (To investigate the effect of PDHK-2 overexpression (PDHK-2/OE) on the lifecycle of 2.0 days . In cont2.0 days . Furtherer stage . We alsodaf-16(mu86) or nhr-49(nr2041) mutants compared to daf-2(e1370) and N2 worms under well-fed conditions, consistent with the absence of a role for PDHK-2 in the context of an active glycolytic pathway.On the basis of the results presented here, we propose the following metabolic axis: PDHK-2 deficiency→decreased lipase-mediated fat consumption→increased survival rate under adverse nutritional conditions . This hypothesized relationship highlights an important regulatory role for PDHK-2 in starvation and the dauer state, where the glycolytic pathway has been shown to be less active C. eleganspdhk-2 expression increased MLS by 20% compared to control worms at 20°C. Our results clearly favor the latter of these two contradictory findings, but only in the survival rate of long-term dauers  in the hope that we can see better some effect of these two factors on the longevity of pdhk-2 mutant in the future.Although PDHK-2 has previously been shown to contribute to longevity in m dauers , in whicpdhk-2 promoter was mainly detected in head neurons  and the intestine  (this report), lipase expression is reduced (through an unknown mechanism), thereby inhibiting fat lipolysis and leading to sustained maintenance of a fat energy source necessary for survival. If PDHK-2 is also defective in dauer stage, lipase activity would also be reduced. In case of daf-2, there was essentially no change in lipase activity between dauer day 1 and day 10 (data not shown), indicating that different types of lipases might become activated during the dauer maintenance period  and decrease fat storage K04A8.5 knockdown partially suppressed the longevity of daf-2(e1370). In mammals, PDK-4 appears to be important in controlling diabetes and obesity C. elegans dauer larvae, fine-tuning of energy consumption is critical to the success of long-term survival through diapause. Similarly, the dauer state of C. elegans is regarded as a hibernating period in which accumulated fats are consumed to support survival throughout this non-aging state. PDHK-2 levels were dramatically increased in this dauer state, consistent with an essential role for PDHK-2 in dauer fat metabolism and survival.PDHK-2 deficiency, which causes the increased survival rate through suppression of lipase activity in dauer larvae, appears to have the opposite effect in non-dauer animals. Loss of germline and reduced daf-2(e1370) mutants leading to abnormal dauer formation through interruption of fat regulation, which is critical for dauer entry , daf-2(e1370), daf-7(e1372) and daf-16(mu86), were obtained from the Caenorhabditis Genetics Center. nhr-49(nr2041) was kindly provided by Carl Johnson and Nemapharm Pharmaceuticals. pdhk-2(tm3075) and pdhk-2(tm3086) were provided by the Japanese National Bioresource Project and were outcrossed six times to a wild-type background. Worms were cultured on nematode growth media (NGM) agar plates seeded with the Escherichia coli strain OP50, as previously described daf-2(e1370);pdhk-2(tm3075) and daf-2(e1370);pdhk-2(tm3086) double mutants were obtained through genetic crosses.The pdhk-2 promoter::green fluorescent protein (GFP) construct (pdhk-2p::GFP) was produced by cloning a 2.5-kb sequence upstream of the pdhk-2 start codon into the pPD95.75 vector using standard molecular biology protocols. The pdhk-2p::PDHK-2::GFP construct contained a 1.2-kb pdhk-2 cDNA sequence (immediately prior to the stop codon) between the promoter sequence and GFP in the pPD95.75 vector. Constructs were microinjected at 50–100 ng/µl into N2, daf-2(e1370), and pdhk-2 mutant worms. GFP-expressing transgenic worms were selected from the progeny and analyzed.The Starvation assays were performed as described previously Total RNA was isolated from worms at various stages using the illustra RNAspin Mini RNA Isolation Kit . Total RNA was reverse transcribed with the Transcriptor First Strand cDNA Synthesis Kit (Roche) using oligo (dT) primers. Quantititative polymerase chain reaction (qPCR) was performed using an MJ Research Chromo4 Detector with the QuantiTect SYBR Green PCR kit, as described by the manufacturer (Qiagen). β-actin was used as an internal control. The data are presented as means (± standard deviations) of triplicate reactions from two or three independent experiments.Lifespan assays were performed at 20°C under well-fed conditions. Embryos from bleached adults were allowed to hatch in S-basal medium to obtain highly synchronous L1 stage animals. L1 worms were then transferred to NGM plates and allowed to mature to the adult stage. Adult worms were transferred to fresh NGM plates each day during the period of reproduction. Worms that no longer responded to gentle prodding with a platinum wire were considered non-viable.Worms were stained with Oil Red O as described previously g and 4°C to remove any insoluble matter. Protein concentration was measured by the Bradford assay. TG content was determined using a commercially available TG determination kit (Sigma-Aldrich). Lipase activity was determined using a QuantiChrom Kit (BioAssay Systems).Proteins from well-fed and dauer worms were harvested as described previously daf-2(e1370), daf-7(e1372) or daf-2;pdhk-2 double mutants) on assay plates (seeded with 2 ml OP50 culture) for 60 hours at 25°C. Dauer larvae thus obtained were washed five times with distilled water and diluted to 500 dauers/10 µl. A 500 µl suspension of dauer larvae was mixed in a 360° rotator (30 rpm) at 20°C so that dauer may continuously consume energy for survival. The five micro liter (5 µl) aliquots of dauer larvae suspension were spotted on the petri dish (>10 spots). Those worms showing L4 stage (exit from the dauer stage) and dead worms were removed from the plate and excluded from worm counting . About 5 µl aliquots of dauer larvae suspension were treated with 1% SDS and survival rate was determined 1 h after SDS treatment. Values represent the means (± SDs) of each test, which was repeated five times. For the statistical analysis, we used student's t-test which has been used previously Dauer worms were obtained by growing synchronized L1 stage worms  is located on chromosome III in C. elegans, and is comprised of 8 exons and 7 introns. The gray boxes denote exons while white boxes, black lines, and dotted lines represent translated regions, introns, and deleted segments, respectively. Mutant tm3075 has a deletion that encompasses regions of exon 6, intron 5, and part of intron 6, which correspond to a portion of the ATPase-like ATP binding region and the histidine kinase motif. Mutant tm3086 has a deletion in exons 3 and 4, as well as and introns 2 and 3, which are translated into mitochondrial branched-chain alpha ketoacid dehydrogenase kinase.(TIF)Click here for additional data file.Figure S2Lifespan and fat content of pdhk-2 mutants under well-fed conditions. (A) Lifespan analysis of N2, pdhk-2(tm3075), and pdhk-2(tm3086) worms. (B) Oil Red O staining in day 1 adults. (C) Triglyceride content in N2, pdhk-2(tm3075), pdhk-2(tm3086), daf-2(e1370), daf-2;pdhk-2(tm3075), and daf-2;pdhk-2(tm3086) worms at day 1 adults. Three independent experiments were performed. Error bars indicate the standard deviation.(TIF)Click here for additional data file.Figure S3Expression pattern of the PDHK-2::GFP in C. elegans. A PDHK-2/GFP fusion construct was microinjected into N2 worms. (A) and (B), GFP-tagged PDHK-2 promoter is shown to be expressed in the head, intestine and the nuclei of intestinal cells. (C) and (D), pdhk-2p::PDHK-2::GFP expressed in the head and intestine. Both constructs were microinjected into worms at 50–100 ng/µl. Scale bar: 50 µm(TIF)Click here for additional data file.Figure S4Expression of PDHK-2 in the head neurons of JK2868 dauer stage worms.pdhk-2 p::PDHK-2::red fluorescent protein constructs were microinjected into JK2868 worms at 50–100 ng/µl. Dauer worms following 7 days of starvation at 25°C. Red indicates pdhk-2 p::PDHK-2::red fluorescent protein and green corresponds to lag-2 p::GFP. Scale bar: 50 µm.(TIF)Click here for additional data file.


CHO (Chinese Hamster Ovary) cells are the cell line of choice for therapeutic protein production. Although the achieved volumetric titers have increased significantly over the past two decades, the establishment of well-producing CHO cell lines is still difficult and not always successful . FactorsCell culture: CHO-DUKX-B11 (ATCC-CRL-9096) and CHO-DG44 (life technologies) were serum-free cultivated in spinner flasks. CHO-K1 (ATCC-CCL-61) and CHO-S (life technologies) were serum-free cultivated in in shaker flasks.E.coli carrying the Rosa 26 BAC (~220 kbp) were transformed with a plasmid coding for a recombinase. Consecutively, a plasmid carrying the gp140 (CN54) gene flanked by homologous regions to the BAC was used for the transformation of the recombinase positive E.coli cells. BAC positive colonies were selected and the BAC DNA was purified .BAC Recombineering: Transfection and selection: CHO-S host cells were transfected with linearized, lipid complexed (Lipofectin) CN54 Rosa26 BAC DNA. Recombinant clone selection was performed in 96-well plates using 0.5 mg/mL G418. BAC transfected CHO cells are able to express the transgene as well as a Neomycin resistance gene within the Rosa26 locus.7 cells/mL) followed by CHO-K1 (8.39 × 106 cells/mL) , ActiCHO (GE-PAA), DMEM/Ham's F12 (Biochrom) + supplements (Polymun Scientific), and CD-DG44 (life technologies) media in spinner and shaker flasks. CHO-DUKX-B11 and CHO-DG44 grew best in spinner flasks with CD-DG44 media, whereas CHO-K1 and CHO-S grew best in shaker flasks with ActiCHO media. The dhfr negative cell lines were growing to much lower viable cell densities than K1 and S. CHO-S reached the highest viable cell density (1.17 × 10CHO-S was chosen for test-transfections and recombinant gp140 (CN54) producers were established using a Rosa 26 BAC construct carrying the gp140 (CN54) gene. The best clone was analyzed in a batch experiment and yielded 77 μg/mL which is ~10 times the titer achieved with a recombinant plasmid derived CHO-DUKX-B11  are at least without a co-transfection of the dhfr gene not growing to high cell concentrations. Rosa 26 BAC derived clones need no amplification as they provide their own open chromatin region. Thus, higher specific productivity can be achieved by elevated transcript levels compared to conventional plasmid clones. The combination of cells growing to high cell densities and the transcriptional efficiency of the Rosa26 BAC system leads to accumulation of significantly increased volumetric titers for a difficult to express glyco-protein.


LIT1-differentially methylated region (MDR) and H19-DMR, exist. A small number of patients with SRS harbor maternal uniparental disomy of chromosome 7q as well. Extensive genetic analyses including methylation specific (MS) PCR-RFLP, MS-MLPA, microsatellite markers or MS-pyrosequencing analysis were performed using genomic DNA obtained from peripheral leukocytes to identify the epigenetic detects in patients with BWS and SRS. Ten out of 14 BWS patients (71.4%) showed hypomethylation in LIT1-DMR. One BWS patient harbored hypermethylation in H19-DMR (7.1%). Two BWS patients had both H19-DMR and LIT1-DMR defects, one of whom has paternal UPD at chr. 11 (14.3%). Eleven out of 13 SRS patients (78.6 %) showed hypomethylation in H19-DMR. One SRS patient (6.7%) had UPD at 7q. With MS-pyrosequencing analysis, epigenetic defects were identified in 93.1% of BWS patients and 85.7% of SRS patients. These positive rates are higher than previously reported positive rates, 80% in BWS and 50% in SRS. In addition, with MS-pyrosequencing analyses, quantification of methylation defects was available, which could identify partial methylation defects that were not revealed by MS-PCR-RFLP or MS-MLPA. The validity of MS-pyrosequencing method for the genetic diagnosis of BWS or SRS is needed to be investigated in a large patient cohort.Beckwith-Wiedemann syndrome (BWS) is fetal overgrowth syndrome, whereas Silver-Russell syndrome (SRS) is characterized by pre- or postnatal growth retardation. BWS and SRS share molecular epigenetic defects in chromosome 11p15, where two imprinting centers,


The potent ribonucleotide reductase (RNR) inhibitor 3-aminopyridine-2-carboxyaldehyde-thiosemicarbazone (3-AP) was tested as a chemosensitizer for restored cisplatin-mediated cytotoxicity in platinum-resistant ovarian cancer.2, day one to four) and cisplatin  infusions every 21 days until disease progression or adverse effects prohibited further therapy. Pre-therapy ovarian cancer tissues were analyzed by immunohistochemistry for RNR subunit expression as an indicator of cisplatin plus 3-AP treatment response.Preclinical in vitro platinum-resistant ovarian cancer cell survival, RNR activity, and DNA damage assays were done after cisplatin or cisplatin plus 3-AP treatments. Six women with platinum-resistant ovarian cancer underwent four-day 3-AP  resolved quickly after cisplatin-alone or 3-AP preceding cisplatin exposure, but persisted after a cisplatin plus 3-AP sequence. On trial, 25 four-day overlapping 3-AP and cisplatin cycles were administered to six women (median 4.2 cycles per patient). 3-AP-related methemoglobinemia (range seven to 10%) occurred in two (33%) of six women, halting trial accrual.2) infusions produced modest methemoglobinemia, the expected consequence of ribonucleotide reductase inhibitors disrupting collateral proteins containing iron.When sequenced cisplatin plus 3-AP, RNR inhibition restored platinum-sensitivity in platinum-resistant ovarian cancers. 3-AP (96 mg/mClinicalTrials.gov NCT00081276 Ovarian cancer is a leading cause of cancer-related mortality in women worldwide, in part due to a greater than 65 percent incidence of intraperitoneal disease persistence, or less than six month disease recurrence after platinum chemotherapy . Chemothde novo production of deoxyribonucleoside triphosphates (dNTP) used in DNA synthesis and repair ) residual 24-hour γH2AX foci. 3-AP treatment alone was associated with no substantial 24-hour residual γH2AX foci (P = 1.0). A sequence of 3-AP then cisplatin provided higher γH2AX foci counts than untreated cells (P < 0.001) or cisplatin-treated cells (P < 0.01). Cells exposed to cisplatin plus 3-AP  showed a significantly high number of retained 24-hour γH2AX foci, as compared to any other treatment. While other mechanisms modulating chemoresistance are likely, 3-AP renders cells more vulnerable to DNA damage when co-administered, rather than given prior to, DNA damaging agents. In-depth time course modeling of γH2AX foci formation and resolution kinetics after DNA damaging agent and RNR inhibitor therapy are underway.To ascertain whether RNR pharmacologic blockade hampered repair of cisplatin-mediated DNA damage and corresponded to observed cytoreduction Figure , manual Characteristics of six women with epithelial ovarian cancer enrolled on GOG protocol #126O are itemized in Table 2) intravenous infusions.In one dyspneic patient, on day 1 of the first cycle of 3-AP, blood oxygen saturation fell to 86% and the methemoglobin level rose to 7% . OxygenP = 0.13), ρ = 0.433 for M2b (p53R2) (P = 0.50) and ρ = 0.233 for M1 (P = 0.70). A partial response was seen in one (17%) of six women whose disease was deemed platinum-refractory. Moreover, stable disease was recorded in two (33%) of six women whose disease was listed as platinum-refractory. The duration of partial or stable disease response was less than six months, and the longest progression-free interval observed was 20 weeks. PFS (median 14 weeks) and OS (median 28 weeks) are displayed in Figure Figure RNR inhibitors have shown high clinical activity and favorable toxicity profiles when co-administered with cytotoxic anticancer therapies, such as cisplatin and radiation ,8. Use ode novo dNTP supply when needed most for cisplatin-DNA adduct repair. Such data mimics radiochemotherapy sensitizing properties of 3-AP in cervix cancer cells [Here, we interrogated whether sequencing 3-AP prior to cisplatin better restored platinum-sensitivity in platinum-resistant ovarian cancer. Our findings that cisplatin plus 3-AP led to substantial DNA damage (i.e. increased number of γH2AX foci), led to impaired RNR activity when dNTPs were most demanded, and led to significant cytoreduction in platinum-resistant ovarian cancer cells are clinically relevant. This is especially noteworthy considering the modest clinical activity seen among the six women with platinum-resistant ovarian cancer treated by an overlapping four-day 3-AP then cisplatin sequence. Sequencing 3-AP, and therefore targeted inhibition of RNR after cisplatin treatment, not only increases cisplatin-mediated DNA damage in “platinum-resistant” ovarian cancer cells, but also blocks er cells ,11. Our de novo dNTPs [The finding of relatively high levels of RNR M2 in non-responders is of interest. RNR M2 is a short-lived protein as a consequence of sequences promoting proteosome-dependent breakdown in late mitosis . It is rvo dNTPs . Current2+-3-AP chelate and of oxygen generating local reactive oxygen species capable of annihilating the nearby tyrosyl free radical. In a similar manner, a Fe2+-3-AP chelate interferes with methemoglobin-hemoglobin cycling. Oxygenated Fe2+ hemoglobin oxidizes to Fe3+ methemoglobin and superoxide at a rate of 3% per day. Methemoglobin is normally reduced to hemoglobin by cytochrome b5 reductase, accounting for 94% of recycling methemoglobin to hemoglobin [Lastly, dose-limiting methemoglobinemia was observed in two women after 3-AP intravenous infusion, halting GOG protocol #126O clinical trial accrual. The mechanism of RNR inhibition by 3-AP is via inactivation of the tyrosyl free radical within the M2 or M2b (p53R2) small subunits ,33,34. Bmoglobin . Methemomoglobin .2) infusions produced modest methemoglobinemia. Pre-clinical studies and phase 1 human trials are needed to determine if RNR inhibitor treatment should be initiated together or promptly after platinum treatment to enhance cytoreduction in other “platinum-resistant” cancers.When sequenced cisplatin plus 3-AP, inhibition of ribonucleotide reductase restored platinum-sensitivity in otherwise platinum-resistant ovarian cancers. 3-AP (96 mg/mRNR: Ribonucleotide reductase; dNTP: Deoxyribonucleoside triphosphates; GOG: Gynecologic Oncology Group; CTCAE: Common Terminology Criteria for Adverse Events; PR: Partial response; S: Stable; PD: Progressive disease; U: Unconfirmed; RECIST: Response Evaluation Criteria in Solid Tumors; IHC: Immunohistochemistry; PFS: Progression-free survival; OS: Overall survival.The authors declare that they have no competing interests.CK carried out the MTT and clonogenic assays, the ribonucleotide reductase activity assay, the γH2AX assay, and drafted this manuscript. TR performed statistical analyses for in vitro assays and immunohistochemistry, and participated in the drafting of this manuscript. FAK carried out the immunohistochemistry, the scoring of staining intensity, and participated in the drafting of this manuscript. JF enrolled patients in the clinical trial and assisted in the drafting of this manuscript. OA recruited patients for clinical trial participation and assisted in the drafting of this manuscript. AB recruited patients for the clinical trial and assisted in the drafting of this manuscript. LU designed the clinical trial, performed toxicity assessments, and helped draft this manuscript. All authors read and approved the final manuscript.


P = 0.0037), and negatively associated with glycaemic control (HbA1c) (P = 0.0035) 3 months after disease onset, should replace this sentence in the abstract of the existing paper: Furthermore, we identified miR-25 as negatively associated with residual beta-cell function , and positively associated with glycaemic control (HbA1c)  3 months after onset.The following sentence: Furthermore, miR-25 one month after diagnosis was positively associated with residual beta-cell function (


Caenorhabditis elegans microRNA-80 (mir-80) is a major regulator of the DR state. mir-80 deletion confers system-wide healthy aging, including maintained cardiac-like and skeletal muscle-like function at advanced age, reduced accumulation of lipofuscin, and extended lifespan, coincident with induction of physiological features of DR. mir-80 expression is generally high under ad lib feeding and low under food limitation, with most striking food-sensitive expression changes in posterior intestine. The acetyltransferase transcription co-factor cbp-1 and interacting transcription factors daf-16/FOXO and heat shock factor-1 hsf-1 are essential for mir-80(Δ) benefits. Candidate miR-80 target sequences within the cbp-1 transcript may confer food-dependent regulation. Under food limitation, lowered miR-80 levels directly or indirectly increase CBP-1 protein levels to engage metabolic loops that promote DR.Caloric/dietary restriction (CR/DR) can promote longevity and protect against age-associated disease across species. The molecular mechanisms coordinating food intake with health-promoting metabolism are thus of significant medical interest. We report that conserved  Caenorhabditis elegans microRNA gene that keeps dietary restriction programs off when food is abundant. A mir-80 deletion exhibits beneficial features of dietary restriction regardless of food availability, including extended maintenance of mobility and cardiac-like muscle function later into life as well as lifespan extension. We identify three key longevity genes that are required for these benefits. We hypothesize that miR-80 is a core regulator by which diverse and intersecting metabolic pathways are coordinately regulated to respond to nutrient availability.Dietary restriction, limitation of calorie intake with maintained vitamin and mineral support, can extend lifespan and protect against diseases of age across many species. Elaboration of molecular mechanisms that control dietary restriction in simple animal models may therefore inform on strategies to activate health-promoting metabolism to help address clinical challenges associated with aging and age-associated disease. We characterize a single  The promotion of healthy aging is a goal of modern medicine, and simple interventions that protect against age-associated decline and disease are the dream of many in the general population. Genetics, environment, and stochastic factors all make substantial and complex contributions to healthspan. Single gene mutations that affect conserved pathways in model organisms can extend life and slow age-associated decline Caenorhabditis elegans, food limitation that results in lifespan extension can be introduced via several protocols eat-2 mutant, whereas with a DR protocol in which bacterial food is diluted on plates, DAF-16/FOXO is essential for lifespan extension In the nematode C. elegansmicroRNAs (miRNAs) can be metabolic regulators C. elegansmir-80(Δ) benefits. Of these, the cbp-1 transcript includes sequences that might be directly targeted by miR-80 to coordinate this circuit. Our data suggest an approach to metabolic activation of DR even under ad lib feeding that could inspire strategies for treating obesity, limiting age-associated disease, and promoting healthy aging.We took advantage of the powerful reagents for miRNA study in C. elegans miRNAs that might impact healthy aging, we screened available C. elegans mir deletion strains mir-80(nDf53) [hereafter referred to as mir-80(Δ)], exhibits low age pigment fluorescence levels late in life compared to wild type (WT) animals (mir-80(Δ) is rescued by a transgene array harboring a mir-80(+) gene, confirming that the low age pigment phenotype is conferred by mir-80 deletion itself. Thus, late in adult life (∼2/3 through the WT lifespan), mir-80(Δ) mutants exhibit low age pigment accumulation typical of healthy aging animals.Our previous studies revealed that age pigment levels (lipofuscin+advanced glycation end products) inversely correlate with locomotory healthspan—low age pigment levels late in life are typical of animals that age gracefully and maintain strong locomotory vigor, whereas high age pigment levels are typical of same-chronological age animals that age poorly and appear decrepit mir-80(Δ) mutants exhibited additional healthspan phenotypes, we next measured two indicators of maintained muscle integrity and function late into adult life–pharyngeal pumping rates and swimming vigor. Pharyngeal pumping is the mechanism by which food is pulled into the gut using specialized cardiac-like muscles. Pharyngeal pumping rates decline markedly with age, such that after the first week of life, feeding capacity is greatly diminished mir-80(Δ) late in life (day 11) as compared to WT (44% increase), a phenotype reversed by a mir-80(+) transgene (mir-80(Δ)  mutants are not simply hyper-activated for pumping, but rather maintain pumping function better late into life. We conclude that mir-80(Δ) exerts a positive effect on the quality of cardiac-like muscle aging.To test if C. elegans body wall muscle deteriorates with age, featuring sarcomere loss mir-80(Δ) to show that mir-80(Δ) mutants are significantly more vigorous swimmers in late adulthood (mir-80(Δ) swim similarly (mir-80(Δ) delays locomotory aging without altering young adult swimming behavior itself.As occurs with human skeletal muscle sarcopenia (the debilitating progressive loss of muscle mass and strength that accompanies aging across species), mir-80(Δ) mutants exhibit several features of extended healthspan, we examined the longevity phenotype. We find that mir-80(Δ) mutants exhibit both mean and maximum healthspan extension, subject to mir-80(+) transgene rescue (mir-80 confers longevity.Given that mir-80(Δ) confers multiple features of extended adult healthspan late in life: lowered intestinal age pigment accumulation, maintained pharyngeal pumping capacity, increased swimming vigor, and lifespan extension. Because mir-80(Δ) does not exhibit notable defects in development  might act via a DR mechanism to extend healthspan and lifespan, we tested mir-80(Δ) mutants for phenotypic features of the DR state  for age pigments at ∼345 nm. However, under all DR-inducing conditions we previously tested  consistently exhibits the DR Exmax shift despite growth in the presence of abundant food (mir-80(Δ) exhibits the spectral signature of DR despite the presence of food, consistent with mir-80(Δ) being a DR constitutive mutant.To ask whether DR state . Our preant food  and has mir-80(Δ) had a somewhat scrawny and pale appearance and on average is ∼10% shorter in length than WT  mutants resembles that of DR animals.WT animals under DR appear thin and pale mir-80(Δ) exhibits a significant extension of reproductive period, producing progeny through Day 8 as compared to WT Day 6, p<0.001, mir-80(Δ) (p<0.05 for Day 3 and p<0.001 for days 4–6), without a significant difference in the total number of surviving progeny (not shown), a pattern of progeny production similar to that of feeding-defective DR mutant eat-2 (mir-80(Δ) exhibits reduced fecundity, similar to animals experiencing DR.Reduced fecundity is associated with DR across species nt eat-2 . We conceat-2 mutant), leads to a reduced lifespan mir-80(Δ) is long-lived relative to WT under normal growth conditions (mir-80(Δ) lifespan is decreased relative to WT in the presence of metformin  is hypersensitive to metformin, consistent with mir-80(Δ) being in a DR constitutive state.The anti-diabetes DR-mimetic drug metformin can induce a life-prolonging DR-like state in WT animals, but administering metformin to animals already in DR ;Is007[skn-1-gfp] reporter strain, whereas this reporter is not highly expressed in wild type Is007[skn-1-gfp] animals that are grown under ad lib feeding conditions (mir-80(Δ) mutant (mir-80(Δ) and 92% eat-2 mutants exhibited strong signals in 1–2 ASI neurons; but 36% of WT only express weak signal in at best one ASI.SKN-1 is a transcription factor crucial for endodermal development and response to oxidative stress nditions . We foun) mutant . At day mir-80(Δ) induces molecular features of DR.Our data support that mir-80(Δ) mutant has a lean body, reduced fecundity, hypersensitivity to metformin, and expresses both molecular and fluorimetric markers of DR despite growth in abundant food. Importantly, unlike the eat-2 mutant, pumping rates in mir-80(Δ) are normal in young animals and are actually enhanced relative to WT later in life (mir-80(Δ) does not physically reduce the ability to eat, but rather is likely to act further downstream to influence DR metabolism. Remarkably, then, deletion of a single miRNA gene can shift C. elegans metabolism into DR to promote healthy aging.In summary, the  in life . Thus, mmir-80::GFP fusion transcriptional reporter . 5/5 extra-chromosomal transgenic lines of this reporter exhibit a broad cellular expression pattern, somewhat similar to the published extrachromosomal array line . The Pmir-80L pattern is distinctive in exhibiting highest expression in the two most anterior gut cells and in posterior gut under high food conditions -regulated DR, we used RNAi to knockdown genes previously implicated in DR lifespan benefits, hypothesizing that genes required for mir-80(Δ) DR should be needed for the Exmax shift and low age pigment levels typical of multiple DR states. Of the 18 genes we screened, we found that RNAi knockdown of transcription factors daf-16/FOXO, heat shock transcription factor hsf-1, and CREB binding protein homolog cbp-1 modulated both the Exmax shift and low age pigment levels of mir-80(Δ) (To identify genes required for ir-80(Δ) ; 6A,B.daf-16/FOXO, an important modulator of longevity through insulin signaling, is also critical for lifespan extension benefits of serial dilution of bacteria on plates (sDR) and peptone dilution on plates (pDR) mir-80(Δ);daf-16(Δ) double mutant was reversed for both the DR Exmax shift . Moreover, the mir-80(Δ);daf-16(Δ) double mutant had a short lifespan, similar to that of daf-16(Δ) (mir-80(Δ) background. Our in silico analyses did not identify candidate miR-80 target sites in the daf-16 transcript, suggesting an indirect role in the mir-80(Δ)-regulated DR pathway.Transcription factor t Day 4, , p<0.05 t Day 4, , p<0.005af-16(Δ) . These dmir-80(Δ) and the insulin signaling pathway further, we compared longevity phenotypes of mir-80(Δ) and mir-80(Δ) treated with daf-2 RNAi, which targets the C. elegans insulin receptor (mir-80(Δ) lifespan can be further extended by daf-2(RNAi) (p<0.005). The additive effects of mir-80(Δ)+daf-2(RNAi) knockdown suggest that healthspan and longevity benefits of mir-80(Δ) may be conferred in part by a daf-2-independent pathway. However, the fact that mir-80(Δ) does not further extend daf-2(RNAi) lifespan (p<.98) is also consistent with a model in which miR-80 partially down-regulates the insulin pathway, and that daf-2(RNAi) reflects a stronger activation of the DAF-16-dependent transcriptional response, more toward an optimal healthspan signaling strength. Regardless of the details of pathway overlap, our data are definitive in establishing that daf-16/FOXO is needed for fluorimetric properties and longevity outcomes of mir-80(Δ).To address the relationship of receptor . We findmax shift that typifies DR . To determine if hsf-1 is also required for mir-80(Δ) longevity, we examined survival curves for the mir-80(Δ);hsf-1(sy441) double mutant. We find that disruption of hsf-1 eliminates the lifespan extension conferred by mir-80(Δ)  . We concir-80(Δ) . In silimax shift ). Thus, cbp-1 activity plays a role in mir-80(Δ) regulation of age pigments, and appears generally needed for mir-80(Δ) DR metabolism. Consistent with a contribution to DR benefits, we find that the lifespan extension conferred by mir-80(Δ) depends strongly on cbp-1 . In the bDR regimen, cbp-1 deficiency has been shown to disrupt expression of daf-16 and hsf-1 target genes p<0.003; .mir-80(Δ) healthspan, cbp-1 is the only one for which the transcript is predicted to include potential miR-80 miRNA target sequences  or both the 5′UTR and the exon 8 candidate binding sites (5+8BS)  .ad lib fed animals +/− mir-80, with a focus on the posterior gut region in which mir-80 regulation is most dramatic. We find that the NBS construct is not regulated by miR-80(Δ)  animals. We measured CBP-1 protein levels using anti-CBP antibodies against human CREBBP for WT and mir-80(Δ) mutants (day 7). We find that CBP-1 protein levels are significantly increased in mir-80(Δ) mutants compared to WT  is associated with increased CBP-1 protein.If  p<0.05, . Thus, icbp-1 is translationally repressed by binding of miR-80 to target sites within the cbp-1 transcript  induces skn-1::GFP expression in the ASI neurons  DR is interesting in multiple regards. First, DAF-16/FOXO and HSF-1 can each individually bind to CBP-1 in nematodes and mammals  blocked expression of DAF-16 and HSF target genes sod-1 and sip-1, respectively, rather than blocking transcriptional induction of daf-16 and hsf-1 that accompanies bDR. These data suggest that the CBP-1 cofactor couples and modifies transcriptional outputs of DAF-16- and HSF-1-dependent longevity pathways under bDR conditions, a model likely to apply for mir-80(Δ)-induced DR.The requirement for mir-80(Δ). For example, knockdown of either AMPK subunit encoded by the C. elegans genome, aak-1 or aak-2, can alter age pigment levels -induced DR benefits. The potential cbp-1 target sites for miR-80 binding are unusual, being situated in the 5′ UTR and within a highly conserved exon. Interestingly, the 5′ UTR sequences in cbp-1 are perfectly conserved in C. brenneri, C. briggsae, and C. remanei (though not in C. japonica) and the exon 8 site is somewhat conserved among all -induced age pigment and lifespan changes. Given that cbp-1 plays a role in dietary restriction associated with growth in axenic medium, growth on diluted bacteria, and eat-2 feeding impairment mir-80 expression regulation under bacterial dilution and dietary deprivation, and a partial engagement of the insulin signaling pathway in mir-80(Δ)-induced longevity  or in the double mir-81(Δ) mir-82(Δ) mutant (data not shown) and thus mir-80 is the sole family member that can be deleted to induce the DR Exmax shift. Interestingly, however, the quadruple mutant mir-80; mir-58; mir-81-82 has a very small body size, more severe than the scrawny body type we documented for mir-80(Δ) (mir-80 high copy number transgene mir-80 family members.The most conserved ir-80(Δ) , which cmir-80 is homologous to the miRNA bantam in Drosophila melanogaster  , none of which have been well studied for function in mammalian biology. The human hCBP coding sequences corresponding to Ceemir-80) . If the C. elegans under standard conditions E. coli strain OP50-1 or HT115 for RNAi. Age-synchronized cultures were prepared by bleach treatment egg preparation, with hatch counted as day 1. Note all presented data represent 3 independent combined trials; error bars +/− SEM. Protocol details are provided in Figure Legends and Supplemental Methods. Note all presented data in this paper represent at least 3 independent combined trials; error bars +/− SEM; we counted age with the egg hatch corresponding to day 0.We grew A detailed list of strains is included as C. elegans under standard conditions E. coli strain OP50-1 or HT115 for RNAi feeding experiments . To generate synchronized cultures, we bleached gravid adults and starved L1 progeny. The wild-type strain was var. Bristol N2 mir-80(nDf53) allele breakpoints are 5′- tgctttcgatgtctatactctc -3′ and 5′-tctggcgaacgaaatgagt-3′, encompassing part of the promoter region, the entire precursor sequence and ∼300 bp downstream. We genotyped mir-80(Δ) by PCR using primer pairs mir80Out-F (5′- ttcgtcgccatcaacacacg-3′)+mir80Out-R (5′- gagcgcggatagatatacagtcag-3′) that flank the deletion and mir80Flank-F (5′- caacaacgatgtgaatgctcgtc-3′)+mir80Flank-R (5′- ctcgcacacggacggactgcc-3′) that bind internal to nDf53. We worked with a 6× outcrossed line. The mir-80 deletion mutant does not exhibit gross developmental phenotypes (mir-80(Δ), supporting that mir-80 does not contribute an essential role in development and basic function. Thus, mir-80 deletion primarily impacts adult maintenance and DR phenotypes.We grew mir-80L:mCherry transcriptional reporter, we amplified the mir-80 promoter using primers 5′-cgagatgagaagtaagaagagtgg-3′ and 5′-tccgtgtgcgagagagtgagcgag-3′ and cloned into the Pmec4::mCherry plasmid vector at the start codon of mCherry from rol-6 co-injection marker (100 ng/ul) to generate extrachromosomal transgenic lines ZB3039-ZB3043.For the Pcbp-1 promoter (4.4 kb until start codon) using primers 5′-gACTAGTc tcttcc atgtcg gtttaa gcgcgg aaacgg tttttt aaa-3′ and 5′-tcccCCCGGGggga caatta gtagaa aaatgt atatat ttgac-3′ containing Spe1 and Xma1 restriction sites, respectively. This product was introduced within the Spe1-Xma1 digested pKS(-) vector. The mir-80 target sites were incorporated within primers that amplified the GFP coding sequence using the primer sites (outlined below) and introduced at the Xma1 site of the above cloned Pcbp-1 vector: NBS: 5′- tccccccgggatgagtaaaggagaagaacttttcactgg-3′+5′-cggggtaccctatagttcatccatgccatgtgtaatccc-3′; 5+8 BS: 5′- tccccccgggaacagctatatctggtgatttgatgagtaaagaagaag-3′+5′-cggggtacctaagatcctcttgactgaacacttcatagttcatccatgcc-3′For the binding site test constructs , we amplet al.max determination at Day 4, we used Datamax software (Horiba Scientific) to identify the peak excitation value. The peak for tryptophan fluorescence was also analyzed to normalize scores, as TRP levels do not change markedly with age.We grew age-synchronized animals (see above) under standard conditions  and scanned animals (n≥50 per strain) for age pigment accumulation  using a Fluorolog 3 spectroflorimeter as in Gerstbrien act-1). Primers used were act1RT-F (5′- ttactctttcaccaccaccgctga-3′) and act1RT-R (5′- tcgtttccgacggtgatgacttgt -3′) for act-1, ama1RT-F (5′- cctacgatgtatcgaggcaaa-3′) and ama1RT-F (5′- cctccctccggtgtaataatg-3′) for ama-1, hsf1RT-F (5′-tagtaatggcagagatgcgtgcga-3′) and hsf1RT-R (5′- tggctgcatgacagagacgagaaa-3′) for hsf-1 and hsp16.2RT-F (5′- atggaacgccaatttgctccagtc-3′) and hsp16.2RT-R (5′- tccttggattgatagcgtacgacc-3′) for hsp-16.2. We plotted Ct values obtained from amplification for target DR genes against this standard curve to determine transcript levels.We synchronized strains by alkaline bleaching We synchronized strains refer  by alkal2EDTA, 1 mM EGTA, 1% NP-40, 1% Sodium deoxycholate, 2.5 mM beta-glycerophosphate, 1 mM Na3VO4)+Protein sample buffer and heated at 95°C for 15 mins. 25 ul of samples was loaded onto a MiniProtean TGX gradient gel  and transferred onto PVDF membrane following separation. Membrane was blocked using 5% non-fat milk in PBST buffer for 1 hour. Membrane was then incubated with CBP-1 and TUB-1 antibodies (Santa Cruz Biotechnology) at 1∶500 and 1∶4000 dilutions respectively in 2% non-fat milk overnight at 4°C. Protein bands were detected using the ECL reagent (Invitrogen) using horseradish peroxidase conjugated secondary antibodies (Jackson ImmunoResearch Labs) at 1∶10,000 dilutions. Band intensities were calculated using ImageJ We synchronized strains by alkaline bleaching Figure S1mir-80(Δ). We grew age-synchronized WT (black), mir-80(Δ) (red) or the mir-80(+) (grey) under standard plate conditions . At day 9, we placed 10 healthy animals per plate, and we scored viability as movement away from pick touch on the indicated days. Statistics were calculated using the OASIS software. Details are presented in Table format. Three additional trials that did not include the rescued strain, as well as other trials featured in the text, showed similar trends, on the order of 10–30% lifespan extension. Note the some trials with mir-80 transgene rescue suggest that overexpression of mir-80 may be deleterious, and that in a small minority of trials, we did not see life extension although the culture always trended in that direction.Individual lifespan data for lifespan analysis of (TIF)Click here for additional data file.Figure S2mir-80(Δ) mutant exhibits hypersensitivity to the DR-mimetic drug metformin, similar to DR mutant eat-2. We grew age-synchronized WT (black), mir-80(Δ) (red) or the eat-2 mutant (blue) under standard plate conditions supplemented with 50 mM metformin . At day 9, we placed 10 healthy animals per plate, ≥40 per strain per trial, and we scored viability as movement away from pick touch on the indicated days. The upper left graph (A) represents data combined from 3 independent trials, which are presented individually in the other panels (B-D). Statistics are calculated using the Log-rank Test. Error bars indicate ± S.E.M. Metformin reduces lifespan for mir-80(Δ) and eat-2 as compared to WT.The (TIF)Click here for additional data file.Figure S3mir-80::GFP reporters are regulated by food availability. mir-80::GFP] line VL211 (wwEx18) grown in the presence of unlimited E. coli. Animals are at day 6 from the hatch for all images in this figure. + indicates anterior. mir-80::GFP] in line VT1492 (maIs196) grown in the presence of unlimited E. coli. White arrow indicates the fluorescent cell that is well labeled in this integrated line, tentatively identified as the excretory duct cell. . Example of expression of Ex[Pmir-80::GFP] in line VL211 grown in the presence of unlimited E. coli to young adulthood and then switched to no food for 48 hours. mir-80::GFP] line VT1492 excretory duct cell (white arrow) grown in the presence of unlimited E. coli to young adulthood and then switched to no food for 48 hours. mir-80 promoter fusion reporter lines in food vs. food limitation. (E) Fluorescence of overall Ex[Pmir-80::GFP] line VL211, (F) excretory duct cell Is[Pmir-80::GFP] line VT1492; after 48 hrs on no-food plates. Food limitation in these studies was by dietary deprivation Published ne VL211 , wwEx18 e VT1492 , maIs196(TIF)Click here for additional data file.Figure S4mir-80 fail to co-localize with ASI sensory neurons, so up-regulation of skn-1::GFP expression in those neurons in DR may be a non-autonomous consequence of miR-80 activity. The expression of transcription factor skn-1 in the two head ASI sensory neurons can be necessary for DR lifespan extension benefits mir-80(Δ) increases the expression of a skn-1::GFP reporter in the ASI neurons . We reared animals under standard conditions . We labelled {A} VL211 expressing Pmir-80::GFP with red DiO, and reciprocally, {B} strain ZB3042 containing bzEx207[Pmir-80L::mCherry] with yellow fluorescent DiI, using a standard protocol that enables the amphid neurons that are open to the environment  to dye-fill. For both approaches, we never observed co-label of the mir-80 reporter with any amphid neurons (n = 30 per reporter); white arrows indicate ASI in representative images. Thus, although mir-80(Δ) influences skn-1::GFP expression in ASI neurons in older animals (mir-80L::mCherry lines Pmec-4::GFP (top row) and Pmir-80::mCherry (bottom row) lines were age-synchronized via alkaline bleaching and plated on standard NGM containing OP50-1 as food. Day 4 animals were imaged using 3 filter sets – DIC (left), FITC  and Texas Red . Touch neurons labeled with GFP in the Pmec-4::GFP strain are clearly and distinctly visible using the FITC channel (white arrows) and are absent in the Texas Red channel. Conversely, mCherry signal from a transcriptional reporter of mir-80 is clearly visible in the TexasRed channel (white arrows) but is absent in the FITC channel. Thus, our images of mCherry in adult intestine is unlikely to inadvertently score age pigment fluorescence.A,B. Two different transcriptional reporters for  neurons . Thus, a animals , express(TIF)Click here for additional data file.Figure S5mir-80 is regulated by food availability under multiple food restriction conditions and as assayed with multiple reporters. We grew strains either under abundant food conditions or using the dietary limitation protocol indicated. Graphs represent measured fluorescence levels from areas indicated for 2 trials of at least 50 animals per DR regimen , measured 48 hours after dietary limitation. Error bars represent Mean Intensity ± S.E.M. Pairwise comparisons were made using Two-tailed Students' T-test, ** - p<0.0005, ** - p<0.005. a) overall pmir-80GFP line VL211 expression, food dilution on solid NGM media. b) overall pmir-80GFP line VL211 expression, food dilution in liquid media. c) excretory duct cell pmir-80GFP line VT1492 expression, food dilution on solid NGM media. d) excretory duct cell pmir-80GFP line VT1492 expression, food dilution in liquid media. e) overall fluorescence pmir-80LmCherry expression for 4 independently derived lines, in abundant food or under dietary deprivation. In individual trials, 4/5 tested lines exhibited significant differences; one line was not regulated in this direction (not shown).(TIF)Click here for additional data file.Figure S6hsp-16.2 transcripts are elevated in mir-80(Δ), but HSF-1 transcript levels are maintained. Temperature-induced HSF-1 activation occurs post-translationally, via formation of an active trimer from inactive monomers that persist under basal conditions mir-80(Δ) mutants exhibit increased HSF-1 transcriptional activity compared to WT, we used qRT-PCR to measure transcript levels of HSF-1 target gene hsp-16.2mir-80. Our data suggest that loss of mir-80 indirectly upregulates HSF-1 activity to increase expression of HSF-1-dependent target genes. A. Transcriptional expression of HSF-1 target gene hsp-16.2 is elevated in mir-80(Δ). We grew age-synchronized WT and mir-80(Δ) under standard conditions of abundant food  and harvested animals at Day 4 for total RNA isolation. We normalized raw qPCR scores for hsp-16.2 (an hsf-1 target) to ama-1. Graphs represent cumulative data from 3 independent trials with 3 technical replicates per trial. Error bars represent ±S.E.M. for RAW transcript gene values normalized to RAW ama-1 levels. Normalized transcript levels of hsp-16.2 are elevated in mir-80(Δ), * p<0.1 (2-tailed Student's T-test). B. hsf-1 transcript levels (day 4) are not changed by the absence of miR-80. We grew age-synchronized WT animals in abundant food and harvested animals at Day 4 for RNA isolation. Raw qPCR scores for hsf-1 were normalized to ama-1. Graphs represent cumulative data from 3 independent trials with 3 technical replicates per trial. Error bars represent ±S.E.M for RAW transcript gene values normalized to RAW ama-1 levels. In Day 4 animals normalized hsf-1 transcript levels were similar in WT and mir-80(Δ) . We noted a trend toward lower hsf-1 transcript levels at day 7 (not shown), which we think reflects modulation of the dynamic transcriptional network altered by miR-80. C. Transcriptional expression of HSF-1 target gene hsp-16.2 is elevated in mir-80(Δ). We grew age-synchronized WT (black bars) and mir-80(Δ) (red bars) under standard conditions of abundant food  and harvested animals at Day 4 for total RNA isolation. We normalized RAW qPCR scores for hsp-16.2 to act-1. Graphs represent cumulative data from 3 independent trials with 3 technical replicates per trial. Error bars represent ±S.E.M for RAW transcript gene values normalized to RAW act-1 levels. Data were compared using 2-tailed Student's T-test. Normalized transcript levels of hsp-16.2 are elevated in mir-80(Δ), * p<0.07. D. hsf-1 transcript levels are not regulated by the presence of miR-80 at day 4. We grew age-synchronized WT (black bars) and mir-80(Δ) (red bars) under standard conditions of abundant food  and harvested animals at Day 4 for RNA isolation. RAW qPCR scores for hsf-1 were normalized to act-1. Graphs represent cumulative data from 3 independent trials with 3 technical replicates per trial. Error bars represent ±S.E.M. for RAW transcript gene values normalized to RAW act-1 levels. Data were compared using 2-tailed Student's T-test. In Day 4 animals normalized hsf-1 transcript levels were similar in WT and mir-80(Δ) (p = 0.14 act-1). We noted a trend toward lower hsf-1 transcript levels at day 7 (not shown) that we think reflects modulation of the dynamic transcriptional network altered by miR-80.Relative HSF-1 target gene (TIF)Click here for additional data file.Figure S7cbp-1 mRNA in the posterior gut, is a conserved miRNA and potential binding sites for miRNAs in human CREBBP are present at sites analogous to those in C. elegans. A. Constructs used to test candidate miR-80 binding sites present in cbp-1 for roles in translational repression. We used the native cbp-1 promoter to express GFP reporters that included i) GFP lacking any candidate miR-80 binding sites (NBS); ii) the 5′ UTR candidate miR-80 binding site and the exon 8 candidate miR-80 binding site from cbp-1 (5+8BS). B. Shown are GFP intensities for extrachromosomal p cbp-1GFP constructs without candidate miR-80 binding sites  or with the 5′ and exon 8 candidate miR-80 binding sites added (5+8BS); in WT (black line) or mir-80(Δ) (red line) backgrounds, day 7, n>30, posterior gut. Animal segments imaged and fluorescence measured as in mir-80 is lacking, whereas the NBS construct is not. The 5+8BS high copy number construct also variably exhibited some “sponge” effects that might be attributed to titrating out endogenous miR-80 and family members (not shown). C. Exon structure of C. elegans cbp-1 is indicated by thick blue bars, introns in thin blue lines (see WormBase for details). The rna22 algorithm cbp-1 within the 5′ UTR and within exon 8. The potential alignments of miR-80 (red) to C. elegans cbp-1 (blue) sequences are indicated. Note that the seed match to the exon 8 region is a perfect 10 bp match for C. elegans, and that the target sequence is conserved to some degree in mouse and human CBP1. However, human miR-136 is even a better match in this region (shown here). D. The predicted miR-80 target sites are conserved in the various Caenorhabditis spp. E. Alignments of miR-80 family members from Drosophila melanogaster (bantam) and human with C. elegans miR-80. Seed region is indicated by black bar; yellow highlight, full conservation; blue highlight, conserved. Note that there are 4 close members of the miR-80 family encoded in the C. elegans genome Click here for additional data file.Table S1hsf-1 and cbp-1 as required for the DR-associated Exmax shift phenotype in the mir-80(Δ) background. We examined the literature for genes experimentally implicated in DR and used RNAi to knock these genes down in the mir-80(Δ) background . Since the Exmax shift is diagnostic of the DR state, we reasoned that genetic interventions that reversed the shift would define genes needed for the mir-80(Δ) DR pathway. P-values were calculated using two-tailed unpaired Students T-test; gene knockdowns that confer statistically significant p-values are in bold.An RNAi screen identifies (PDF)Click here for additional data file.Table S2mir-80(Δ) background identify genes required for the low age pigment level phenotype. We used RNAi to knockdown genes previously implicated in DR in the mir-80(Δ) background  and quantitated age pigment levels relative to endogenous Trp levels. Since low age pigment/Trp ratios (AGE/TRP) are associated with the DR state mir-80(Δ) DR pathway and/or for enhanced healthspan. P-values were calculated using the Unpaired two-tailed Student's T-test, values within the 90% confidence level are bold, increase or decrease indicated by arrows.RNAi directed against known DR genes in the (PDF)Click here for additional data file.Table S3Strains used in this study.(PDF)Click here for additional data file.Table S4Summary of lifespan experiments. Median lifespan is calculated from the GraphPad Prism Software while Maximum lifespan was calculated by the mean lifespan of the oldest 10% cohort in each experiment. P-values are calculated from Log-Rank Tests for the entire population for the complete lifespan.(PDF)Click here for additional data file.Text S1Supplementary methods.(PDF)Click here for additional data file.Text S2References used exclusively in the supplemental information.(PDF)Click here for additional data file.


In vitro assays revealed the diagnostic domain of SM7L produced robust photon emission, while the therapeutic domain showed marked anti-tumor efficacy and significant modulation of p38MAPK and ERK pathways. In vivo, the unique multi-functional nature of SM7L allowed simultaneous real-time monitoring of both SM7L delivery and anti-tumor efficacy. Utilizing engineered stem cells as novel delivery vehicles for SM7L therapy (SC-SM7L), we demonstrate that SC-SM7L significantly improved pharmacokinetics and attenuated progression of established peripheral and intracranial human GBM xenografts. Furthermore, SC-SM7L anti-tumor efficacy was augmented in vitro and in vivo by concurrent activation of caspase-mediated apoptosis induced by adjuvant SC-mediated S-TRAIL delivery. Collectively, these studies define a promising new approach to treating highly aggressive cancers, including GBM, using the optimized therapeutic molecule SM7L.Creating new molecules that simultaneously enhance tumor cell killing and permit diagnostic tracking is vital to overcoming the limitations rendering current therapeutic regimens for terminal cancers ineffective. Accordingly, we investigated the efficacy of an innovative new multi-functional targeted anti-cancer molecule, SM7L, using models of the lethal brain tumor Glioblastoma multiforme (GBM). Designed using predictive computer modeling, SM7L incorporates the therapeutic activity of the promising anti-tumor cytokine MDA-7/IL-24, an enhanced secretory domain, and diagnostic domain for non-invasive tracking.  Successful management of many terminal cancer types has not been achieved primarily because many conventional anti-cancer therapies lack tumor specificity and exhibit poor or inadequate pharmacokinetics that result in high toxicity to normal tissue, limited bioavailability, and subsequently ineffective tumor cell killing. Recently, the development of multifunctional therapeutics offers unprecedented potential to overcome the limitations of current anti-cancer therapies in vitro and in vivoTo date, the majority of multifunctional agents are synthetic nanoscale devices or nanoparticles chemically engineered with tumor targeting and/or imaging components in vitro and in vivo. SM7L was designed to incorporate: 1) the anti-tumor domain of MDA-7/IL-24 to introduce cancer cell-specificity and robust therapeutic activity, 2) light-emitting luciferase domain to impart diagnostic imaging capacity, and 3) altered secretion sequence to optimize extracellular delivery. Our results demonstrate the multifunctional properties of SM7L and the efficacy of stem cell-mediated SM7L therapy against highly aggressive brain tumor GBM. These results define a promising new approach to treating highly aggressive cancers using the multifunctional anti-cancer molecule SM7L.In the current study, we engineered and validated a new multifunctional molecule SM7L, and tested the anti-tumor efficacy of stem cell-delivered SM7L therapy using models of the deadly brain cancer GBM both Predictive molecular modeling was used to interrogate the structure of wild type-MDA-7/IL-24, and provide a rational basis for designing a potentially optimized molecule. The “bystander” antitumor effects of MDA-7/IL-24 protein are mediated by binding to the IL-20R1/IL-20R2 and IL-22R1/IL-20R2 (IL-20Rα/β) receptors  on the sTo allow easy and efficient transduction of cells with SM7L, we next engineered lentiviral vectors encoding wild-type MDA-7/IL-24 (M7) and the optimized SM7L . To asseIn vitro BLI showed a direct correlation between mNSC-SM7L cell number and photon emission  were engineered with LV encoding SM7L (mNSC-SM7L), wild-type MDA-7/IL-24 (mNSC-M7), or control (mNSC-GFP), and transduction was confirmed by fluorescence imaging . In vitremission , while lemission . To deteemission , and theemission . Westernemission . Taken tWe next determined which events in stem cell-mediated delivery to tumors could be tracked by a combination of multimodality imaging and diagnostic markers utilizing the additional diagnostic properties of SM7L. To this end, GFP-labeled stem cells secreting SM7L were implanted adjacent to established U87 glioma cells expressing mCherry-FLuc in skin fold window chambers . High-rein vivo pharmacokinetics and therapeutic efficacy of SM7L delivered by engineered stem cells. In the clinic setting, therapeutic stem cells are typically injected directly into tumors. When mNSC-SM7L were implanted around established U87-FLuc tumors to mimic clinical cell engrafts typical of stem cell-based treatments, bioluminescence imaging revealed a concentrated and stable SM7L signal that co-localized with the established tumor and showed no change in levels through 24 h . To investigate GBM therapy via activation of multiple anti-tumor pathways, U87  and U251 . U87-FLuc human GBM cells were implanted intracranially in mice with control mNSC-GFP, mNSC-SM7L, mNSC-S-TRAIL, or the double secreting mNSC-SM7L/S-TRAIL. Serial Fluc imaging revealed mNSC-SM7L/S-TRAIL induced a significant reduction in tumor volume that persisted through 4 weeks of treatment , cDNA encoding the 60 bp signal sequence of Flt-3 ligand was amplified with primers including Nhe1 and BamH1 restriction sites using previously described DNA as template BamH1 and Xho1 restriction sites. The two fragments were digested and ligated into CSCGW plasmid digested Nhe1/Xho1. To engineer the luciferase fusion variants, the cDNA encoding Gaussia luciferase (Gluc) was amplified using the pGluc-Basic vector  as a template with primers containing EcoV and Xho1 restriction sites. Both LV-SM7 and LV-M7 vectors contained an EcoV site in the c-terminus of MDA-7/IL-24. Both vectors were digested with EcoV and Xho1, and the Gluc fragments were ligated in-frame. LV-Fluc-mCherry was kindly provided by Dr. Andrew Kung . Lentiviral vectors were produced by transient transfection of 293 T cells as previously described The lentiviral transfer plasmid CS-CGW Human glioblastoma cell lines U87-Fluc  Guide for the Care and Use of Laboratory Animals, USDA regulations, and the American Veterinary Medical Association: 1) To determine the effects of stem cell-delivered SM7L on intracranial GBMs, 2×105 U87-Fluc human glioma cells and 1×105 mNSC expressing SM7L or control (GFP-Rluc) were implanted stereotactically into the right frontal lobe of SCID mice . On days 1,3, 6, 8, 14, 22, 29, and 35 post-injection mice received an intraperitoneal injection of D-luciferin  and tumor progression was monitored by FLuc imaging. On day 29 post-implantation, a subset of mice from control (mNSC-GFP-Rluc) and mNSC-SM7L treatment groups were injected with coelenterazine (100 µg) and Rluc or Gluc imaging was performed to assess stem cell volumes or SM7L levels respectively. 2) To determine the effects of stem cell-based SM7L/S-TRAIL combination therapy on intracranial GBMs, 2×105 U87-Fluc human glioma cells and 1×105 mNSC expressing SM7L, S-TRAIL, SM7L/S-TRAIL or control (GFP-Rluc) were implanted stereotactically into the right frontal lobe of SCID mice . Tumor progression was then monitored by FLuc imaging on days 1 and 29 as described above. All the other methods utilized in this study are described in detail in In this study, several tumor models were utilized. In all studies, a 4 µl injection volume was used for tumor cells and a 6 µl injection volume was used for stem cells. All experimental protocols were approved by The Subcommittee on Research Animal Care at Massachusetts General Hospital, and care of the mice was in accordance with the standards set forth by the National Institutes of Health Data are expressed as mean±SD and were analyzed by either Student’s T-test or ANOVA (after Bartlett’s test of homogeneity of variance), followed by the Newman-Keuls correction for multiple comparisons. Differences were considered significant at P<0.05.Figure S1Levels of IL20α/β receptors in GBM cell lines. (A) Relative expression of IL20α/β receptors on various GBM determined by RT-PCR. Experiments were performed in triplicate with mean and SD reported.(TIF)Click here for additional data file.Figure S2In vitro characterization of stem cell-delivered SM7L on glioma line, U251. (A) Representative images and summary graphs of cell viability assays performed on U251 established human GBM cells co-cultured with mNSC-GFP, mNSC-M7, or mNSC-SM7L. U251-Fluc-mcherry GBM cells were seeded in 96 well plates. 24 hrs later, control or mNSC secreting wild-type or SM7L were overlayed on the cells. Three and five days later, Fluc imaging was performed to determine the effects of stem cell-delivered M7 or SM7L on GBM cell growth. (B) Summary data demonstrating the viability of GBM8 primary patient-derived cells co-culutred with mNSC-GFP or mNSC-SM7L. Cell viability was determined by Fluc imaging 1, 3, and 5 days post-treatment. *p<0.05 vs. control. Experiments were performed in triplicate with mean and SD reported.(TIF)Click here for additional data file.Methods S1in vitro and in vivo imaging.This section includes details on LV transduction, molecular modeling, caspase and viability assays, in vitro bioluminescence imaging and fluorescence microscopy, western blot analysis, quantitative RT-PCR, tumor models and (DOC)Click here for additional data file.