5 mg on two occasions: 7 days prior to dosing with GLPG0259 (on day -7) and on day 14 at the same time as the last GLPG0259 dose. GLPG0259 free base (10 mg/mL in 40% [w/v] hydroxypropyl-ß–cyclodextrin, pH 3) or a matching placebo was administered once daily for 14 days, using a syringe, as for study 1. Subjects in the 50 mg dose group were additionally administered an oral dose of methotrexate 7.5 mg (3 tablets of Ledertrexate® 2.5 mg; Wyeth-Pfizer) on two occasions. A dose of 4 mg of folic acid (Folavit®; Kela Pharma Selleckchem MM-102 NV) was administered 24 hours after each methotrexate administration as a preventive measure for methotrexate toxicity. Folic acid was administered after all safety and pharmacokinetic

assessments had been done. Blood samples for pharmacokinetics were collected at regular intervals over 24 hours (on days 1 and 13 [in the 50 mg cohort only]) or over 7 days after the last dose on day 14 (i.e. up to day 21) to assess plasma concentrations of GLPG0259. Blood check details sample handling was similar to that described for study 1. For methotrexate (in the 50 mg cohort only), CH5424802 blood samples were collected at regular intervals over

24 hours (on day -7 and day 14) in tubes containing lithium heparinate, in order to obtain plasma, and were stored at -20°C until analysis. Study 3: Oral Relative Bioavailability and the Food Effect This was a phase I, open-label, randomized, three-period, three-treatment crossover study to compare the oral bioavailability of a solid dosage form of GLPG0259 (a capsule) relative to an oral solution, and to evaluate the effect of food on oral bioavailability of GLPG0259 formulated as a capsule in healthy subjects (n = 12). The criteria for subject eligibility were the same as those listed for study 1. The treatments consisted of an oral dose of a 50 mg GLPG0259 free-base solution given after an overnight fast (treatment A), a GLPG0259 fumarate capsule (equivalent to 50 mg free base) given after an overnight fast (treatment B), and a GLPG0259

fumarate capsule (equivalent to 50 mg free base) given 30 minutes after the start of Etomidate a high-fat, high-calorie breakfast (treatment C). Each subject was administered treatments A, B, and C in one of the two treatment sequences (i.e. ABC or ACB) determined by a computer-generated randomization schedule. There was at least a 7-day washout period between treatments for each subject. Subjects were admitted to the clinical unit on the evening prior to dosing (day -1) and were confined until 24 hours after the last dose. For treatment A, GLPG0259 free base was administered as 5 mL of 10 mg/mL in 40% (w/v) hydroxypropyl-ß–cyclodextrin (pH 3), using a syringe. A volume of 235 mL of water was given to each subject immediately at the time of dosing. Capsules to be administered for treatments B and C were filled with 50 mg of GLPG0259 as a fumarate salt.

Thus, endocrine therapy may play a role in treating hormone-dependent cancers by decreasing the metastases that are caused by MMP7 activation. To test this hypothesis, ARS-1620 mw we examined the ability of TAM to decrease MMP7 activation in the ERβ-positive colon cancer cell line HT29. Methods

Cell culture and treatment HT-29 cells are highly metastatic colon carcinoma cells that were obtained from the American Type Culture Collection, Rockville, MD, USA. Cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum at 37°C in a humidified atmosphere of 5% CO2. Drug administration schedules TAM and fluorouracil (5-FU) were purchased from Sigma (St Louis, MO). The drug-exposure PX-478 ic50 schedules, which are summarized in Table 1, were as follows: (a) no treatment; (b) TAM alone (1 × 10-7, 1 × 10-6, 1 × 10-5, or 1 × 10-4 M) for 48 h; (c) 5-FU alone (6.25, 12.5, 25, or 50 μM) for 72 h; (d) 12.5 μM 5-FU for 24 h followed by 12.5 μM 5-FU plus indicated TAM for 48 h. The experiments were performed in triplicate for each time point, and the means ± SD were calculated. Appropriate amounts of drug solution were added directly to the growth

medium the day after plating. Control cells were plated in growth medium supplemented with 0.1% DMSO. Table 1 Schedule of each group of treatment for three different times Group 24 h 48 h 72 h (a) no treatment     (b) TAM TAM   (c) 5-FU 5-FU 5-FU (d) 5-FU 5-FU+TAM 5-FU+TAM Drug sensitivity, as indicated by the MTT assay To induce cell death, cells were treated with either TAM (Sigma, Cat. No. T-9262) dissolved in DMSO or 5-FU. The final concentrations ranged from 1 × 10-7 to 1 × 10-4 M for click here TAM and from 6.25 to 50 μM for 5-FU. To test the www.selleckchem.com/products/H-89-dihydrochloride.html Cytotoxicity of each drug, HT-29 cells in the exponential growth phase were seeded into 96-well cell plates

in 100 μl of culture medium for 24 h prior to drug exposure and then treated with various concentrations of TAM, 5-FU, or a combination of these drugs. Cytotoxicity was evaluated using a tetrazolium-based semi-automated colorimetric (MTT) assay, with an ELISA reader at OD490. Flow cytometry analysis HT-29 cells were seeded in 6-well plates at a density of 4 × 106 cell/well. Cells were treated with various concentrations of each drug for the appropriate times, incubated at 37°C, fixed in 70% ethanol, and labeled with propidium iodide solution (50 μg/ml; Sigma-Aldrich). The DNA content and cell cycle distribution of approximately 1 × 106 stained cells were analyzed using a FACScan flow cytometer (Becton Dickinson). Reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA was isolated from 4 × 106 cells by TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA was reverse transcribed in a total volume of 20 μl containing 2 μg RNA, 0.5 μg olig (dT)15, and 15 μl DEPC-treated water. Reverse transcription reaction was incubated at 30°C for 10 min, 48°C for 30 min, and 99°C for 5 min.

Plates were incubated at room temperature (25°C) for 1 h to allow bacteria to attach to the skin. Following incubation, the Idasanutlin clinical trial suspension was vacuumed from each well, and skin sections were gently washed with distilled water and vacuumed to remove unattached bacteria. This washing process was repeated once more. After removal of excess solution, initial bioluminescence on skin sections was quantified for 15 s of exposure selleckchem using the IVIS imaging system. One mL of 4°C distilled water was added to each well of the appropriate plate for each serotype. The other plate for each serotype received one

mL of 25°C distilled water. The plate that received 4°C distilled water remained at refrigeration temperature (4°C) for 2 h on a rotating stage at 200 rpm. The plate that received 25°C distilled water remained at room temperature (25°C) for 2 h on a rotating stage at 200 rpm. At the conclusion of the 2 h washing period, water was Nirogacestat mw vacuumed from each well, and bioluminescence from bacteria attached to the chicken skin was measured at 37°C for 5 min. The total flux of bioluminescence from each well was divided by the corresponding bacterial density value of the original bacterial suspension to normalize bioluminescent flux. Acknowledgements We thank Dr. Alain

Givaudan (INRA, Université Montpellier II, Montpellier, FRANCE) for providing us with Photorhabdus luminescens genomic DNA. We acknowledge Dr. Scott Willard and Dr. Peter Ryan for use of the IVIS Living Image System in the MSU Laboratory for Organismal and Cellular Imaging. This study was funded by the U.S. Department of Agriculture, Agricultural Research Service (agreement no 321956-182070-027000-371290). References 1. Ohl ME, Miller SI: Salmonella : a model for bacterial pathogenesis. Annu Etofibrate Rev Med 2001, 52:259–274.PubMedCrossRef 2. Ly KT, Casanova JE: Mechanisms of Salmonella entry into host cells.

Cell Microbiol 2007, 9:2103–2111.PubMedCrossRef 3. Sarlin LL, Barnhart ET, Caldwell DJ, Moore RW, Byrd JA, Caldwell DY, Corrier DE, Deloach JR, Hargis BM: Evaluation of alternative sampling methods for Salmonella critical control point determination at broiler processing. Poult Sci 1998, 77:1253–1257.PubMed 4. Lillard HS: Incidence and recovery of Salmonellae and other bacteria from commercially processed poultry carcasses at selected pre- and post-evisceration steps. J Food Prot 1989, 52:88–91. 5. Lillard HS: The impact with commercial processing procedures on the bacterial contamination and cross-contamination of broiler carcasses. J Food Prot 1990, 53:202–204. 6. Lillard HS: Bacterial cell characteristics and conditions influencing Salmonella adhesion to poultry skin. J Food Prot 1985, 48:803–807. 7.

tuberculosis H37Rv. ABC transporter proteins are found in both eukaryotes and prokaryotes and constitute a large super family of multi-subunit permeases that transport various molecules (ions, amino acids, click here peptides, antibiotics, polysaccharides, proteins, etc.) across biological membranes, with a relative specificity for a given substrate [43]. They consist of two hydrophobic membrane spanning domains (MSDs) associated with two cytoplasmic

nucleotide binding domains (NBDs) [44–46]. They are classified as importers and exporters depending on the direction of translocation of their substrate [47]. Importers are found exclusively in prokaryotes and are involved in the uptake of extracellular molecules [48]. Exporters are found in both prokaryotes and eukaryotes, where they export molecules from the cytoplasm [49]. Taken together, the observation of three transporter proteins with higher PRN1371 supplier abundance in M. tuberculosis H37Rv may suggest a significant role of these proteins in the overall transport of nutrition by the bacilli, influencing its chances for survival, rendering the two strains, although highly similar, in different physiological

states that make one of them more fit for survival in host cells and consequently more pathogenic. On the other hand, 10 membrane-associated proteins were observed with >5x or higher relative abundance in M. tuberculosis H37Ra. Only three of those (Rv0014c, Rv0070 and Rv1030), were proposed to have a biological function, the role of the rest is yet to be determined. The gene encoding transmembrane serine/threonine-protein kinase pknB (Rv0014c) protein was found to be essential for mycobacterial growth. This protein is thought to be involved in signal transduction via phosphorylation. PknB has been shown to be a substrate for phosphoserine/threonine phosphatase PstP (Rv0018c), which is also up-regulated in M. tuberculosis H37Ra, and its kinase activity is affected by PstP -mediated dephosphorylation. PknB and phosphoserine/threonine phosphatase PstP (Rv0018c) may act as a Etofibrate functional pair in vivo to control mycobacterial cell growth [50, 51].

The putative gene GlyA2 (Rv0070) has been proposed to encode for the enzyme buy AZD1390 serine hydroxymethyltransferase (SHMT), up-regulated in M. tuberculosis H37Ra, is a pyridoxyl 5- phosphate (PLP)-dependent enzyme. The SHMT reaction plays a major role in cell physiology as it is considered to be a key enzyme in the pathway for interconversion of folate coenzymes that provide almost exclusively one-carbon fragments for the biosynthesis of a variety of end products such as DNA, RNA, ubiquinone, methionine, etc. [52]. The physiological role of SHMT is the reversible interconversion of serine to glycine. From the genome analysis of M. tuberculosis, there is an additional SHMT gene (GlyA1, Rv1093); the relative abundance of this enzyme is similar in both strains.

The optical anisotropy are considered in this paper, and we have studied ϵ 2(ω) under parallel polarization only, which is named as ϵ 2(ω)p. In Figure 5a, the pure (8,0) ZnO nanotubes have four peaks located at about 2.6, 8.3, 11.1, and 15.0 eV. The first peak located at 2.6 eV is mainly due to the transition from O 2p states

to Zn 4s states. The second peak at 8.3 eV corresponds to transitions between the Zn 3d Selleck GF120918 states and O 2p states. The peaks at 11.1 and 15.0 eV are associated with the electron transition between Zn 3d states and O 2s states. For the Ag1 configuration, the peak in the range from 5.0- to 13.0-eV energy region originates from the Zn 3d states to O 2p states and BIBF 1120 chemical structure Zn 3d states to O 2s states. The peak in the low-energy region at about 0.1 eV mainly comes from the electronic interband transition between Ag 4d states and Zn 4s states in the conduction band. The peak positions of the Ag1N2, Ag1N2,3,4, and Ag1N3,4 configurations are similar to that of Ag1 configuration

except that the peaks are more intense because of higher N concentration. The peak at about 2.0 eV originates from the electronic transition from Ag 4d states to Zn 4s states for Ag1 configuration while it originates from the electronic transition from Ag 4d to N 2p for Ag1N2, Ag1N2,3,4, Ag1N3,4, Ag1N5, and Ag1N6 configurations. A red shift occurred for the peak at about 0.5- to 2.0-eV energy region for the Ag1N2, GSK2245840 concentration Ag1N2,3,4, (-)-p-Bromotetramisole Oxalate and Ag1N3,4 configurations with the increase of N concentration, because the electron transition energy from the occupied impurity states

to CBM has a red shift, and the gap of the occupied impurity states to CBM are 0.395, 0.366, and 0.201 eV, respectively. Figure 5b shows the dielectric function spectra of Ag1N2, Ag1N5, and Ag1N6 configurations. In Figure 5b, the peak at 1.0- to 5.0-eV energy regions has a red shift, and the volume of the peak increases with the increasing distance of Ag atom and N atom. Figure 5 Dielectric function spectra of pure and Ag-N-codoped (8,0) ZnO nanotubes. (a) Configurations of Ag1, Ag1N2, Ag1N2,3,4, and Ag1N3,4. (b) Configurations of Ag1N2, Ag1N5, and Ag1N6. Figure 6 shows the reflectivity and absorption spectra of pure and Ag-N-codoped (8,0) ZnO nanotubes. For the reflectivity of the pure ZnO nanotube, four peaks (located at 2.5, 6.0, 8.0, and 11.6 eV, respectively) can be observed, which correspond to the ones at 2.6, 8.3, 11.1, and 15.0 eV in ϵ 2(ω), respectively. For the Ag1 configuration, there is a new transition peak near the Fermi energy levels because Ag is doped into the ZnO nanotube, and it is associated with the electron transition between Ag 4d states and O 2s states. However, the peak at about 2.

The transfers from plate to flask were repeated every 3–4 weeks. Selleckchem Nec-1s anaerobic nitrate turnover The capability of An-4 to reduce nitrate anaerobically was investigated in two experiments: (1) An-4 was cultivated in Erlenmeyer flasks under oxic vs. anoxic

conditions in the presence of both NO3 – and NH4 +, and (2) An-4 was pre-cultivated in Erlenmeyer flasks under oxic conditions in the presence of 15NO3 – and then exposed to anoxic conditions in gas-tight incubation vials. In Experiment 1, the fate of NO3 – and NH4 + added to the liquid media was followed during aerobic and anaerobic cultivation of An-4. Six replicate selleck liquid cultures were prepared P005091 ic50 as described above, but with the YMG broth adjusted to nominal concentrations of 50 μmol L-1 NO3 – and 50 μmol L-1 NH4 + using aseptic NaNO3 and NH4Cl stock solutions, respectively. Three cultures

were incubated aerobically, whereas the other three cultures were incubated anaerobically by flushing the Erlenmeyer flasks with dinitrogen for 30 min and then closing them with butyl rubber stoppers. Subsamples of the liquid media (1.5 mL) were taken after defined time intervals using aseptic techniques. Anaerobic cultures were sampled in an argon-flushed glove box to avoid intrusion of O2 into the Erlenmeyer flasks. Samples were immediately frozen at −20°C for later analysis of NO3 – and NH4 + concentrations. In Experiment 2, the precursors, intermediates, and end products of dissimilatory nitrate reduction by An-4 were investigated in a 15N-labeling experiment, involving an oxic-anoxic shift imposed on axenic mycelia. For the aerobic pre-cultivation,

a liquid culture was prepared as described above, but with the YMG broth Amylase adjusted to 120 μmol L-1 15NO3 – (98 atom% 15N; Sigma-Aldrich). For anaerobic incubation, fungal aggregates were transferred to gas-tight glass vials (5.9-mL exetainers; Labco, Wycombe, UK) filled with anoxic NaCl solution (2%) amended with nitrate as electron acceptor and glucose as electron donor. Using aseptic techniques, equally-sized subsamples of fungal aggregates were transferred from the aerobic pre-cultures into 30 replicate exetainers. The wet weight of the aggregates was determined. Then the exetainers were filled with anoxic NaCl solution adjusted to 120 μmol L-1 15NO3 – and 25 μmol L-1 glucose. Care was taken not to entrap any gas bubbles when the exetainers were closed with the septum cap. The exetainers were fixed in a rack that was continuously rotated to keep the aggregates in suspension and were incubated at 26°C in the dark for 24 days. The anaerobic incubation was terminated in batches of three exetainers after defined time intervals.

Genetic experiments indicated that this change in cell size homeostasis involves production of the alarmone (p)ppGpp (guanosine-penta/tetra-phosphate), a signaling compound that is a key player of a cellular response to amino acid starvation known as stringent response. Results and Discussion

Our rationale here is that we can get insights into the AZD2014 clinical trial biological role of YgjD by following the cellular response of its depletion on the single cell level and with high temporal selleck compound resolution. We diluted cultures of the conditional lethal P ara -ygjD mutant TB80 onto pads of solid LB medium that either contained L-arabinose (inducing ygjD expression) or D-glucose (repressing

ygjD expression) and used time-lapse microscopy to follow single cells growing into microcolonies, taking an image every 2 or 4 minutes. The images were analyzed with the software “”Schnitzcell”" [18]. The growth rate and cellular morphology of the P ara -ygjD strain grown in the presence of L-arabinose was similar to the wild type grown under the same conditions (Figure 1a and 1c, and Additional file 1 – movie 1 and Additional file 2 – movie 2). Figure 1 ygjD -expression determines patterns click here of growth. Each panel depicts data of cell numbers versus time from three independent experiments; each experiment is based on a microcolony that was initiated with a single cell, and followed over about six Metformin cost to seven divisions. A) TB80 (Para-ygjD) grown in presence of 0.1% L-arabinose. B). TB80 (Para-ygjD) grown in presence of 0.4% glucose. Note that the growth rate decreased after about

150 minutes. C) MG1655 (E. coli wild type) grown in LB medium with additional 0.4% glucose. Growth rates are similar to panel A, indicating that the induction of ygjD-expression in TB80 (panel A) lead to growth rates that are similar to wild type E. coli. A shift of the P ara -ygjD strain to glucose lead to the depletion of YgjD. This depletion is based on two effects. First, transcription of ygjD stops after the shift to glucose. Residual L-arabinose that remains in the cells from growth under permissive conditions is rapidly metabolized. Lack of L-arabinose turns the transcriptional activator (AraC) of the Para promoter into a transcription repressor. In addition, glucose metabolism causes depletion of the cellular co-inducer cyclic AMP. Together these effects lead to effective repression of ygjD transcription in TB80. After termination of de novo ygjD mRNA synthesis the amount of YgjD in each cell declines, because the mRNA and the protein are diluted through cell division, and degraded by cellular nucleases and proteases, respectively [20].

It has also been suggested that

there might be other angiogenic factors, different from VEGF, which are important in testis tumor biology [37]. No significant association was found between VD and VEGF expression or prognosis according to disease-free survival. This could be a consequence of the low recurrence rate in our population (70% of our patients presented a good international risk), making it AZD8931 manufacturer difficult to find a statistical association. With similar results, in a study of 51 patients with stage I disease, no association was found between VD and VEGF expression and DFS [37]. Concerning these results, there is a possibility that angiogenic factors other than VEGF are relevant in the development of this neoplasm’s vascularization, taking into account the fact that modulation of the angiopoietin family has been previously described in non-tumor models [38, 39], as well as fibroblast selleck chemicals Danusertib growth factor [40], metalloprotease

induction, and cellular adhesion-molecule expression [41]. Unexpectedly, we found no correlation between hCG serum levels and VEGF tissue expression. Our results indicate that hCG and VEGF may operate through different signaling pathways for angiogenesis stimulation, and suggest that hCG is not only an independent prognostic factor, but that also it additionally plays a role in the pathophysiology of these neoplasms, representing a potential therapeutic target in patients showing significant elevations of this hormone and who display no response to treatment. Conclusion Our study shows that hCG elevation is independently associated with high VD in testicular germ cell tumors, but not with VEGF expression. This suggests that hCG plays an important function in the angiogenesis and pathophysiology of germ cell neoplasms, being a likely target of treatment by receptor inhibition, activity blockage, or obstruction of intracellular pathways it triggers. References 1. Bosl GJ, Motzer RJ: Testicular germ-cell cancer. N Engl

J Med 1997, 337: 242–254.CrossRefPubMed 2. Boyle P: Testicular cancer: the challenge for cancer control. Lancet Oncol 2004, 5: 56–61.CrossRefPubMed 3. van Basten JP, Schrafford Koops H, Sleijfer DT, Pras E, van Driel MF, Hoekstra HJ: Current concepts about testicular cancer. Eur J Surg Oncol 1997, 23: 354–360.CrossRefPubMed 4. Gori S, Porrozzi S, Roila F, Gatta G, De Giorgi U, Marangolo M: Germ cell tumours of the testis. Thalidomide Crit Rev Oncol Hematol 2005, 53: 141–164.CrossRefPubMed 5. Jones RH, Vasey PA: Testicular cancer: Part 1, Management of early disease. Lancet Oncol 2003, 4: 730–777.CrossRefPubMed 6. Scardino PT, Cox HD, Waldmann TA, Mcintire KR, Mittenmeyer B, Javadpour N: The value of serum tumor markers in the staging and prognosis of germ cell tumors of the testis. J Urol 1977, 118 (6) : 994–999.PubMed 7. Doherty AP, Bower M, Christmas TJ: The role of tumour markers in the diagnosis and treatment of testicular germ cell cancer. Br J Urol 1997, 79 (2) : 247–252.PubMed 8.

3, scheme A. Since the find more iron-restricted growth of S. SB202190 in vitro aureus Δsfa sbnA::Tc and S. aureus Δsfa sbnB::Tc mutants was restored in the presence of L-Dap, we hypothesized that this was due to the mutants’ renewed ability to synthesize staphyloferrin B. To verify this, we performed a chrome azurol S (CAS) assay on concentrated and methanol-extracted culture supernatants of several mutant derivatives of S. aureus Δsfa (grown under iron starvation) to quantify their

siderophore production (Figure 2B and 2C). Consistent with the growth phenotype illustrated in Figure 2A, amendment of growth media with L-Dap allowed siderophore production by S. aureus Δsfa sbnA::Tc and Δsfa sbnB::Tc (Figure 2C). Interestingly, supplementation

of the parental strain (Δsfa) with L-Dap enhanced the level of staphyloferrin B output by approximately five-fold (Figure 2C cf. Figure 2B). As a final method to demonstrate that the siderophore Savolitinib in vitro secreted by S. aureus Δsfa sbnA::Tc or Δsfa sbnB::Tc mutants, in media supplemented with L-Dap, was indeed staphyloferrin B, we performed plate-disk growth promotion assays by spotting culture supernatants onto sterile paper disks that were then placed onto TMS agar seeded with various S. aureus siderophore transport mutants (Figure 2D). Only culture supernatants from S. aureus sbnA::Tc or sbnB::Tc mutants that were fed L-Dap promoted the growth of seeded S. aureus Δhts and its isogenic wild-type strain, but strains containing a mutation in the sirA gene (encoding the receptor lipoprotein for staphyloferrin B) did not grow. Moreover, no growth-promoting siderophore was produced by sbnA or sbnB mutants grown in media Celecoxib lacking L-Dap (Figure 2D). LC-ESI-MS/MS was used for confirmation of staphyloferrin B presence in methanol-extracted culture

supernatants of complemented mutants (data not shown); spectra were as published previously [17]. When iron-restricted growth media were supplemented with several other molecules that were predicted substrates or byproducts of an SbnA-SbnB reaction (e.g. L-ornithine, L-proline, and O-acetyl-L-serine) according to the models illustrated in Figure 3, scheme A, we noted that none rescued the iron-restricted growth of sbnA or sbnB mutants in the Δsfa background (Figure 2E). This leads us to conclude that none of these molecules can be modified into L-Dap by alternative S. aureus enzymes. Figure 3 Proposed schemes for SbnA- and SbnB-dependent synthesis of L-Dap. Scheme A is adapted from Thomas et al. [18] for which the functions of SbnA and SbnB are analogous to the proposed functions VioB and VioK, respectively. The proposed functions of SbnA in schemes B-D remain as a β-replacement enzyme while SbnB is proposed to be an NAD+-dependent dehydrogenase of the indicated amino acid.

We speculated that the respiring cells suspended in spent media containing large amounts of Vactosertib price D-lactic acid were converting this fermentative product into energy-rich metabolites, fueling proliferation and other cellular functions. To test whether the D-lactic acid in the spent media does supply

fuel for growth, we suspended overnight cultures of GD1:pAHG cells in either their own spent media, LB media or the spent media from GD1 cells. We found that the cells provided the GD1 spent media grew nearly as well as cells in LB media, whereas cells suspended in their own spent media showed negligible growth (Figure 5C). These results suggested that respiring E. coli cells utilize D-lactic acid and other metabolites present in the spent media as fuel for proliferation. Under these conditions, the utilization of D-lactic acid has a negative impact on worm life span (Figure 5B). Q deficient E. coli replicate more slowly than wild-type or ATP synthase mutant E. coli Bacteria use a large proportion

of available energy for replication; the loss of Q should lead to slow proliferation compared to wild-type cells. Bacterial proliferation inside the worm is known to influence life span [14]. The ATP synthase mutant strain AN120 has wild-type Q levels but is incapable of utilizing the proton-motive force to produce ATP [33]. The life span extension in worms fed AN120 is similar to that of worms fed an MDV3100 E. coli mutant (1100Δbc) harboring a ZD1839 clinical trial deletion of the entire operon encoding ATP synthase [18]. Worms fed the E. coli parental strain 1100 had life spans indistinguishable from either OP50 or AN180 (the parent strain of AN120) [18]. Life spans of N2 worms fed rescued GD1 (GD1:pAHG) or OP50 are also indistinguishable [17]. Thus we determined the growth dynamics of representative bacterial strains known to influence life span. GD1 E. coli grow more slowly as compared to either OP50 or AN180 and also reach saturation at lower cell density (Figure 6). The AN120 mutant cells show an intermediate rate of growth and cell density at saturation (Figure 6). The bacterial proliferation

observed is consistent with the hypothesis that worms fed diets of the slower growing E. coli strains have longer life span. Figure 6 GD1 E. coli proliferate more slowly than either Cell press wild-type or ATP synthase mutant E. coli. Overnight cultures of the indicated E. coli strains were adjusted to an optical density (A600 nm) of 0.1 in LB medium containing the appropriate antibiotic. The increase in cell number was assayed over time. Solid grey line with open squares, GD1; dotted grey line with +, AN120 (ATP synthase mutant); solid black line with open squares, OP50; dotted black line with X, AN180 (wild-type parental strain of AN120). Asterisks indicate p-value < 0.05 when compared with A600nm of OP50 culture at the 5 and 25 h time points.