The power of mouse embryonic stem (ES) cells to colonise the developing embryo has revolutionised mammalian developmental genetics and stem cell research. This power is vulnerable, however, to the cell culture environment, deficiencies in which can lead to cellular heterogeneity, adaptive phenotypes, epigenetic aberrations and genetic abnormalities. Here, we provide detailed methodologies for derivation, propagation, genetic modification and primary differentiation of ES cells in 2i or 2i+LIF media without serum or undefined serum substitutes. Implemented diligently, these procedures minimise variability and deviation, thereby improving the efficiency, reproducibility and biological validity of ES cell experimentation. Summary: Detailed protocols for the propagation, manipulation and differentiation of mouse embryonic stem cells, with tips for minimising variability and improving the efficiency, reproducibility and reliability of embryonic stem cell culture. Mouse embryonic stem (ES) cells are cell lines derived from the pre-implantation epiblast of mouse embryos . Under aES cells were first derived in 1981 by culturing early mouse embryos in conditions optimised for teratocarcinoma stem cells . The capThe original culture conditions for ES cells comprised co-culture with a feeder layer of mitotically arrested embryonic fibroblasts and medium containing carefully screened foetal calf serum (FCS) . This efin vitro, ES cells are inherently liable to acquire genetic and epigenetic adaptations or abnormalities if subjected to untoward selective pressures. Vulnerability to environmental stress may be enhanced in serum-free culture. Consequently, apparently minor variations in media formulation or culture procedures may give rise to discrepant findings. Reports of subpopulations and cell cycle heterogeneity in 2i culture Wash medium 2i or 2i/LIF [hereafter referred to as 2i (±LIF)] in N2B27  or other culture medium, wash medium and Accutase to 37°C. Note: avoid leaving 2i medium or Accutase at 37°C for too long.Remove all gelatine from plates or wells and replace with warm medium. Return to the incubator to pre-equilibrate . Drying the plates is not necessary.Aspirate medium gently but completely from cells and add 0.5 ml of Accutase per well. Avoid drying the cells.Incubate with Accutase see  at room Add 1 ml of wash buffer and pipette up and down (without touching the bottom of the dish) 10-20 times in order to obtain a single cell suspension. Try to minimise the formation of bubbles. Tip: check under the microscope to ensure single cell suspension.Transfer cell/Accutase/wash suspension to a Falcon tube containing 6 ml of wash medium see .g for 3.5 min.Centrifuge cells at 300 Aspirate supernatant with care, removing as much liquid as possible without disturbing the pellet.Re-suspend cell pellet by pipetting up and down 10-15 times in 0.5-2 ml of 2i (±LIF) medium, ensuring a single cell suspension is obtained.Count number of cells/ml and plate appropriate number of cells see . Slide pCell density will have a significant effect on metabolism, cell cycle and differentiation kinetics, amongst other factors. This is, therefore, a key parameter to monitor. We have observed that culture at high density or colony overgrowth can compromise ability to differentiate and that this effect may become irreversible. Overgrowth of cells in FCS/LIF conditions is also associated with impaired capacity of differentiation and karyotypic instability, but in defined conditions ES cells must be passaged at smaller colony sizes than in FCS- or KSR-containing medium. A single period of overgrowth may induce a permanent change, even if undifferentiated morphology is retained. Specifically, even if overgrown cells regain refractile domed morphology after passaging, they may be compromised. Two key parameters should be kept relatively constant: (1) the density at which cells are plated after each split; (2) the frequency with which cultures are split. Representative images of cultures ready to be passaged are shown in 4 cells/cm2; for most experiments: 1.5×104 cells/cm2 (see below for differentiation). See also The following cell concentrations are routinely used: for maintenance of cells, 1.5-3.0×10In routine culture, cells should be split every 2-3 days . HealthyIf passaging is delayed beyond 2 days, medium should be renewed on day 3, and any day thereafter.The quality of N2B27 must be monitored. Signs of suboptimal N2B27 include flattening of colonies, cells detaching, reduced proliferation rate, or increased cell death.Etv4/5 gene deletions), are sensitive to MEK inhibition and show more robust proliferation in CH/LIF (LIF containing CHIR99021) (ES cells from certain genetic backgrounds (e.g.C57BL/6) require 2i/LIF in order to be stably propagated long term, whereas others, such as 129 strains, can be propagated in 2i alone or in single inhibitor with LIF. LIF invariably increases colony formation after low-density plating. The addition of LIF alters the kinetics of differentiation, however, delaying the process by ∼12 h . CertainIR99021) . To convCell lines can either be derived in defined conditions (see below) or adapted from conventional FCS/LIF (with or without feeders) conditions. When converting cells from FCS/LIF, we recommend plating cells first in FCS/LIF and changing media to 2i or 2i/LIF after 24 h. Cell death, mainly associated with elimination of differentiating cells, is often observed for the first few days, but stable homogeneous cultures can be established within one or two passages.Cells should be routinely maintained without any antibiotics, in keeping with good tissue culture practice . AntibioMycoplasma testing should be carried out frequently and on all newly generated or obtained lines using PCR assays and commercial kits. If mycoplasma is detected, the cultures should be discarded .It is important to maintain two types of cell line stock. Primary stocks are the lowest passage cells available [passage (p) 4-8 for embryo-derived cell lines], that have been genotyped, mycoplasma screened, have at least ∼80% diploid cells (40XY or 40XX) and show uniform morphology. These are kept in liquid nitrogen for long-term storage. Secondary stocks are more numerous at slightly higher passage number (p10-15). These stocks can be kept short term at −80°C and are used for routine experiments. Cells should not normally be passaged beyond 30. However, we have generated high contribution chimeras that give germline transmission after multiple passages of mouse ES cells (p30+), embryonic germ cells (p15+), and haploid ES cells (p20+) in 2i/LIF.Table S1) to ensure complete inactivation, or doubling the volume of wash medium, or increasing the BSA concentration.Accutase is routinely used because it is gentler than trypsin, results in a single cell suspension and does not require serum inactivation. TrypLE reagents may also be used, although these occasionally result in incomplete dissociation. Trypsin can be used, but we recommend adding ∼5% FCS to the wash medium , can be improved by plating onto laminin-coated plates. For this, coat plates with laminin solution (∼10 µg/ml in PBS) for a minimum of 2 h at 37°C, before aspirating the solution and adding the culture medium.2. We use 7% CO2 for compatibility with FCS cultures maintained in GMEM. In the presence of B27 we have not observed any advantage of low O2 compared with ambient atmosphere.DMEM/F12 and Neurobasal buffer can be used at 5-10% COThe colony-formation assay is a key functional test in ES cell biology. Cultures are dissociated to a single cell suspension and plated at very low  density, and the number of colonies that form is determined after several days. This assay can be used as a diagnostic tool for media quality control, and experimentally for determining the proportion of undifferentiated cells in a differentiation or reprogramming study or following a genetic/chemical perturbation, or in a modified culture environment. See Accutase10 µg/ml laminin in PBSWash mediumTable S1 for media formulation and suggested suppliers)2i/LIF  with laminin (∼10 µg/ml in PBS) for a minimum of 2 h at 37°C. At clonal density, laminin helps colonies remain attached for the duration of the experiment.Pre-warm Accutase, wash medium, and culture medium (e.g. 2i/LIF).Before starting, aspirate laminin and add 1 ml of 2i/LIF per well. Return plate to the incubator in the meantime to equilibrate medium.Aspirate medium from cells and quickly add appropriate volume of Accutase.Incubate with 1 volume of Accutase at room temperature (RT) for 4-6 min (1 min after the colonies have detached).Add 1 ml of wash medium and pipette up and down 10-20 times in order to obtain a single cell suspension. Tip: check under the microscope to ensure single cell suspension.Transfer cell/Accutase/wash suspension to a Falcon tube containing 5 volumes of wash medium.g for 3.5 min.Centrifuge cells at 300 Aspirate supernatant with care, removing as much liquid as possible without disturbing the pellet.Re-suspend cell pellet in 0.5-2 ml of culture medium (e.g. 2i/LIF).Count number of cells/ml.Plate 400 cells/well in prepared medium. If necessary, carry out two sequential 1:10 dilutions of the cell to improve accuracy, and aim to pipette ∼50 µl of cell suspension per well.Slide plate back and forth across a flat surface to distribute cells evenly, then place carefully in incubator.After 4-5 days, perform alkaline phosphatase staining according to manufacturer's instructions, let dry, image plate and count the number of colonies.The use of 12-well or 6-well plates is advisable. Plate 400 cells in 12-well plate, and 800 in 6-well plates. It is recommended to perform at least three technical replicates per condition as high variability can occur when plating small numbers of cells. Fluorescence-activated cell sorting of cells directly onto plates can also be used to reduce technical variability.Colonies can detach easily; therefore, add fixative gently. Leaving cells for >5 days will increase the likelihood of colony detachment.We also recommend performing colony-formation assays in parallel FCS/LIF medium and analysing plates by alkaline phosphatase staining on day 3/4. Monitor cultures to make sure colonies do not merge. In FCS/LIF conditions, differentiating cells, which would not survive in 2i/LIF, will persist and contribute to mixed or wholly alkaline phosphatase-negative colonies. This experiment can serve as a control for plating efficiency and differentiation potential. Typically, we expect wild-type cells maintained in 2i or 2i/LIF to have a clonogenic capacity of ∼70-80% when plated in 2i/LIF in optimal media  with minimal differentiation.Examining the kinetics of exit from naïve pluripotency can inform whether a particular perturbation  accelerates, delays or blocks transition to differentiation Wash medium2i (±LIF) in N2B27Table S1)N2B27 . Use a separate plate for each time point.Pre-warm necessary volume of 2i (±LIF) or other culture medium, wash medium and Accutase to 37°C. Note: avoid leaving 2i medium or Accutase at 37°C for too long.Remove gelatine from plates or wells and replace with warm medium. Return to the incubator to pre-equilibrate .Split cells as indicated previously, re-suspend cell pellet in 0.5-2 ml of 2i (±LIF) and count number of cells/ml.Plate 30,000 cells per well of a 24-well plate. Slide plate back and forth across flat surface to distribute cells evenly, then place carefully in incubator. Do not disturb for several hours.After 12-24 h, aspirate medium from cells and add 0.5-1 ml of sterile PBS. Aspirate PBS and replace with pre-warmed N2B27. Perform media replacement steps gently to avoid detaching cells.Analyse cells at the appropriate time points.The exact time at which 2i(±LIF) is replaced by N2B27 to initiate exit from naïve pluripotency is flexible. However, it should be kept constant across experiments. Higher density cultures transition more slowly.Different downstream tests can be performed to determine the kinetics of transition and different time points can be chosen , the batch of N2B27, cell plating density, etc.Monolayer neural differentiation is a simple and well-characterised system  and, undAccutase∼10 µg/ml laminin in PBSWash medium2i (±LIF)Table S1)N2B27  overnight  or for a minimum 2 h at 37°C.Aspirate laminin (do not wash) and add appropriate volume of N2B27. Return to the incubator to pre-equilibrate. Note: pre-equilibration is not necessary but it helps survival.Split cells, re-suspend in N2B27 and count them.4 cells/cm2 or 1.2×104 cells/cm2 for flow-sorted cells. Optional: adding 1 µg/ml laminin directly to N2B27, before plating cells in laminin-coated plates, can help with adhesion.Plate directly in N2B27 onto laminin-coated plate at a density of 1.0×10Change media to fresh N2B27 on day 2 and every day thereafter.Coat plates with laminin (∼10 µg/ml in PBS) overnight at 37°C.Aspirate laminin (do not wash) and add appropriate volume of 2i (+LIF). Return to the incubator to pre-equilibrate. Note: pre-equilibration is not necessary but it helps survival.4 cells/cm2 or 1.2×104 cells/cm2 for sorted cells. Optional: adding 1 µg/ml laminin directly into N2B27 can help with adhesion.Split cells as late as possible in the day and plate in 2i or 2i/LIF onto laminin-coated plate at a density of 1.0×10As early as possible on the next day, gently wash cells with PBS before changing the media to N2B27.Change media to fresh N2B27 on day 2 and every day thereafter.4 cells/cm2, 1.0×104 cells/cm2 and 1.5×104 cells/cm2), monitoring cell death at day 3-4 and quantifying Sox1-positive cells at day 4-5. Efficient conditions typically yield ∼90% Sox1-positive cultures on day 5 with little non-neural differentiation .The quality of N2B27 should be monitored. Batches should be specifically tested for neural differentiation. Certain mouse strains benefit from high insulin N2B27  and in persistence of undifferentiated ES cells.Failure to change media regularly, or plating cell density higher than 1.2×10CRISPR/Cas9  allows fAccutase0.1-0.2% gelatineWash mediumFuGenegRNA vectorsFCS Table S1 for media formulation and suggested suppliers)N2B27, 3 µM CHIR99021, LIF Puromycin (depending on the strategy)HaemocytometerTissue culture treated 3.5 cm diameter plates or 12-well plates and 96-well plates for single clone pickingFalcon tubesCentrifuge2 and 37°CHumidified incubator at 7% COCell sorter (depending on the strategy)Pre-warm CL, wash medium and Accutase at 37°C. Allow FuGene to equilibrate at RT.Coat 3.5 cm diameter, low-edge dish or wells of a 12-well plate with 0.1-0.2% gelatine and place in incubator for a minimum of 15 min.Prepare transfection mixes as follows. Mix A: 250 ng gRNA 1, 250 ng gRNA 2, 200 μl CH/LIF (control: 200 μl CH/LIF); Mix B: 1 μl FuGene, 200 μl CH/LIF .Split cells as previously indicated.Just before counting cells, combine mix A and B to obtain 400 µl transfection reaction. Incubate for 5-15 min at RT (longer incubation might reduce transfection efficiency).Count cells.Aspirate gelatine from wells and plate 300,000 cells in 1.6 ml of CH/LIF.Add 400 µl of transfection reaction to cells and mix.Ensure even distribution of cells by sliding the dish across a flat surface vertically and horizontally.The day after transfection, change media and add CH/LIF+1 µg/ml puromycin.48 h after transfection, change medium to CH/LIF+0.5 µg/ml puromycin for a further 24 h. This should kill all cells in the control plate.72 h after transfection, change medium to CH/LIF+PenStrep.Day 6-8 after transfection, pick clones for expansion. The control plate should be almost completely clear. Colony number per plate ranges from 30 to 100+ depending on the gRNA.The day after transfection, change media and add fresh CH/LIF.48 h after transfection, sort single GFP-positive cells into a 96-well plate in CH/LIF+PenStrep.72 h after transfection, change medium to fresh CH/LIF+PenStrep.62987]. The distance between the gRNA target sequences should be kept <200 bp for efficient insertion.Design gRNAs and clone into nickase plasmid  or rat serum ∼10 µg/ml laminin in PBSAccutaseDissecting microscopeLaminar flow hood2 and 37°CHumidified incubator at 7% COMouth pipettePasteur pipettesOrgan culture dishesTissue culture plasticsPre-equilibrate organ culture dishes containing KSOM (or Blast) +1 µM PDO325901 and 3 µM CHIR99021 (2i/KSOM) in the incubator for at least 15 min. Put PBS in the outer well to prevent evaporation. Flush embryos from oviducts using M2 medium at the 8-cell stage A and plaPrepare and pre-equilibrate a fresh organ culture dish containing 2i/LIF in N2B27  in the central well and PBS in the outer well.Transfer embryos (now at blastocyst stage) to the new dish.Incubate for 1 or 2 more days, depending on when the embryos were transferred to N2B27. A total of 3 days in culture is optimal C.On day 3 of culture, pre-equilibrate an organ culture dish containing N2B27+20% anti-mouse serum and three dishes containing 400 µl N2B27 in the incubator. Also, gelatinise a 96-well plate. After 20 min, aspirate gelatine and add 200 µl 2i/LIF in N2B27. Leave in the incubator to pre-equilibrate.If the embryos have not hatched, remove the zona pellucida by use of acid Tyrode's solution D.Place a drop (∼300 µl) of Tyrode's solution on a sterile flat dish.Transfer embryos still contained within the zona pellucida to Tyrode's solution with minimal carry over and monitor under a dissection microscope until the zona has been dissolved.Once the zona is dissolved, wash embryos in one of the dishes of N2B27.Place embryos in a pre-equilibrated dish containing N2B27+20% anti-mouse serum.Incubate for 30 min to an hour or so in the incubator.Rinse three times in pre-equilibrated N2B27  by transferring embryos through drops.Add 100 µl of freshly thawed rat serum or guinea pig complement to pre-equilibrated 400 µl N2B27 to obtain a 20% solution E,F. IncuNote: it is essential that the time between thawing and use is kept to a minimum because the complement is highly unstable.Place each isolated ICM into a well of the 96-well plate with pre-equilibrated 2i/LIF in N2B27.Incubate for 3-7 days, during which time each ICM will form an ES-like colony G.To passage primary outgrowths, gently aspirate media from wells, add 50µl Accutase per well of 96-well plate and incubate for ∼5 min until primary colonies detach. Add 100µl of 2i/LIF, pipette up and down to obtain single cell suspension, and transfer all content to a new laminin-coated well of a 96-well plate. Next day, change medium to fresh 2i/LIF. Repeat passaging procedure, progressively expanding the line to 48-well, 24-well, etc. At the 24-well stage, passage lines as indicated in ‘Propagation of ES cells without serum factors or feeders’ section.Three main tests are carried out on batches of N2B27 to determine suitability for cell culture:Culture cells in batches of 2i/LIF in N2B27 side-by-side for two or three passages.For testing, coat a 12-well plate with laminin (∼10 µg/ml in PBS), three wells per batch.Plate 400 cells/well in 2i/LIF+batch N2B27.After 5 days, perform alkaline phosphatase staining according to manufacturer's instructions and count the number of colonies.Good batches result in >80% clonogenicity.See ‘Colony-formation assay’ section for a detailed protocol.Culture Rex1::GFPd2 cells for two or three passages in 2i batch-N2B27.Analyse by flow cytometry.Good batches result in log-normal distributions.Note: Other cell lines can be used but if the reporter protein is not destabilised  cells have to be cultured for longer periods. If reporter cells are not available, immunostaining for Nanog can also be performed. Good batches result in relatively uniform Nanog expression across all cells.Follow neural differentiation protocol above and examine cells on day 3-4.Good batches of N2B27 show little cell death until cells reach confluence. Death on day 2-3 is evident in poor batches.Although most batches will enable self-renewal of ES cells, differentiation requires specific attention.Some useful troubleshooting tips are presented in In this paper, we have aimed to provide an accessible and comprehensive set of step-by-step protocols, including all media formulations, to facilitate robust and standardised manipulation of mouse ES cells in defined conditions. We provide guidelines for quality control and troubleshooting tips. The indicated reporter cell lines for calibration are available from the authors. We highlight key parameters that may perturb ES cell behaviour and genetic integrity. In particular, we emphasise the requirement for timely passaging to avoid overgrowth of colonies. Feedback on these protocols and recommendations for further improvement are welcome.


POLG and POLG2, respectively. The accessory subunits enhance DNA binding and promote processive DNA synthesis of the holoenzyme. Mutations in either POLG or POLG2 are linked to disease and adversely affect maintenance of the mitochondrial genome, resulting in depletion, deletions and/or point mutations in mtDNA. A homozygous mutation located at Chr17: 62492543G>A in POLG2, resulting in R182W substitution in p55, was previously identified to cause mtDNA depletion and fatal hepatic liver failure. Here we characterize this homozygous R182W p55 mutation using in vivo cultured cell models and in vitro biochemical assessments. Compared to control fibroblasts, homozygous R182W p55 primary dermal fibroblasts exhibit a two-fold slower doubling time, reduced mtDNA copy number and reduced levels of POLG and POLG2 transcripts correlating with the reported disease state. Expression of R182W p55 in HEK293 cells impairs oxidative-phosphorylation. Biochemically, R182W p55 displays DNA binding and association with p140 similar to WT p55. R182W p55 mimics the ability of WT p55 to stimulate primer extension, support steady-state nucleotide incorporation, and suppress the exonuclease function of Pol γin vitro. However, R182W p55 has severe defects in protein stability as determined by differential scanning fluorimetry and in stimulating function as determined by thermal inactivation. These data demonstrate that the Chr17: 62492543G>A mutation in POLG2, R182W p55, severely impairs stability of the accessory subunit and is the likely cause of the disease phenotype.Mutations in mitochondrial DNA (mtDNA) have been linked to a variety of metabolic, neurological and muscular diseases which can present at any time throughout life. MtDNA is replicated by DNA polymerase gamma (Pol γ), twinkle helicase and mitochondrial single-stranded binding protein (mtSSB). The Pol γ holoenzyme is a heterotrimer consisting of the p140 catalytic subunit and a p55 homodimeric accessory subunit encoded by the nuclear genes  Mitochondria are cellular organelles in eukaryotic organisms responsible for producing ATP via electron transport and oxidative phosphorylation. Human mitochondria have their own genome comprised of a 16.5 kb circular DNA (mtDNA). Hundreds to thousands of copies of mtDNA exist within a cell depending on cell type and growth stage . The regPOLG and POLG2, respectively. P55 has been characterized as a DNA binding protein that enhances and regulates p140  dTTP, 50 μg/mL poly(rA)•oligo(dT)12-18, 220 mM NaCl, varying concentrations of dTTP  and 5 μL of holoenzyme were incubated at 37°C for 9 min. Reactions were stopped by the addition of 1 mg/mL BSA in 0.1 M NaPPi (0.2 mL) and cold 10% TCA (1 mL). The mixture was filtered through Whatman GF/C filters, washed with 1N HCl, rinsed with 100% ethanol then dried. TCA-insoluble reactivity was determined by liquid scintillation counting. Maximal polymerase activity for WT and R182W p55 was set at 100% and apparent Kd(p55) values were derived by nonlinear regression analysis of DNA polymerase activity at each concentration of p55 dimer, as described  dTTP, 50 μg/mL poly(rA)•oligo(dT)12-18, 220 mM NaCl, varying concentrations of dTTP  and a fixed concentration of holoenzyme (1 nM p140 + 2 nM p55) were incubated at 37°C for 0, 3, 6, 8 and 10 min. Reactions were stopped by the addition of 1 mg/mL BSA in 0.1 M NaPPi (0.2 mL) and cold 10% TCA (1 mL). The samples were then filtered through Whatman GF/C filters, washed with 1 N HCl, rinsed with 100% ethanol and dried before TCA-insoluble radioactivity was determined by liquid scintillation counting. Steady-state kinetic values were determined as previously described -dTTP, 50 μg/mL poly(rA)•oligo(dT)12-18 primer-template and 5 μL heat-treated holoenzyme were incubated at 37°C for 10 min. Reactions were terminated and processed as described above. Activity observed for reactions without heat treatment was set to 100%, and remaining activity was plotted against time of 75°C pre-incubation. Data were fit to an equation for exponential decay (Nt = Noeλt-), which permitted calculation of half-lives for inactivating p55 function (t1/2 = ln2/λ).WT and R182W p55 were diluted to 2 nM in EDB on ice and aliquoted into 10 μL samples. WT p55 was pre-incubated at 75°C for 0, 1.0, 3.0, 5.0, 7.0, 9.0, 12.0, 15.0, 25.0, 30.0, 45.0, 55.0, 65.0, 75.0, 90.0, 105.0, 120.0, 135.0, 150.0, 165.0, and 180.0 min while R182W p55 was pre-incubated at 75°C for 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.5, 10.0, 15.0, and 20.0 min. Each time point was placed on ice until the entire series was completed. Addition of 1 nM p140 while still on ice permitted assembly of holoenzymes. Reactions (50 μL) containing 25 mM HEPES-KOH , 2.5 mM 2-ME, 0.5 mM MnClPOLG2 (R182W p55) on cellular health and metabolism, growth rates of patient dermal fibroblasts were assessed in media with glucose or galactose. Requiring cultured cells to utilize galactose as a sole carbon source obligates the cell to rely on oxidative phosphorylation rather than glycolysis for energy production providing a simple test for severe respiratory chain defects [To begin to understand the effects of the mutation at Chr17: 62492543G>A in  defects –39. WhenPOLG2 is essential for mtDNA replication, loss of function mutations in POLG2 may cause a failure of mtDNA replication. The reduced growth rate for patient fibroblasts led us to determine mtDNA copy number to help establish whether R182W p55 is affecting mtDNA replication. Also, we had previously observed that the patient had reduced mtDNA copy number in liver and muscle  [ND1 and ND4 amplification levels (mtDNA) were normalized to nuclear RPPH1 levels to determine relative mtDNA copy number, and standard curves were used to calculate absolute mtDNA copy numbers. R182W p55 patient fibroblasts showed a significant decrease in mtDNA copy number with ND1 being 61% of WT and ND4 being 64% of WT  (Since ctively) . MtDNA c T-test) .Reduced mtDNA copy number could indicate either an inability of R182W p55 to stimulate processive DNA synthesis and/or a reduction in the level of p55 expressed within the cell. To help understand p55 function in the patient fibroblasts, we determined mRNA transcript levels of both p140 and p55 by quantitative real-time PCR. Transcript levels for both p140 and p55 were significantly reduced in glucose  . This inp ≤ 0.05 as determined by Students t-test, The patient fibroblasts showed an inability to produce mtDNA, presumably due to the R182W p55 mutation. However, these cells may have other unidentified mutations within either the nuclear or mitochondrial DNA. To confirm the observations in the patient fibroblast were caused by the R182W POLG2 mutation we episomally expressed either WT or R182W p55 in HEK293 cells. HEK293 cells were transfected with plasmids expressing WT p55 or R182W p55, and analysis of transcripts indicated a 5-fold overexpression of WT p55 compared to endogenous p55 in untransfected cells, and expression of R182W p55 was equivalent to endogenous p55 in untransfected cells. We have previously shown that overexpression of WT p55 does not alter bioenergetics in HEK293 cells . We did E. coli and purified to homogeneity using the established protocol [The analysis of HEK293 cells expressing R182W p55 indicated that the R182W mutation can cause a defect in mitochondrial respiration. To better understand how the R182W p55 amino acid substitution affects biochemical function, the mutation was generated by site-directed mutagenesis, the recombinant protein was overproduced in protocol  with a sP55 binds double strand DNA (dsDNA) independently of p140 , and dimPOLG2 have been shown to disrupt association of p55 and p140 [12-18 to assess the DNA polymerase activity of Pol γ holoenzyme under conditions of saturating dTTP and a restrictive salt concentration. At 220 mM NaCl, the DNA polymerase activity of p140 is insignificant without proper association with p55. Accordingly, p140 activity was assessed at varied and increasing concentrations of p55, and the formation of active holoenzyme is a measure of physical association of p140 and p55 subunits. The inter-subunit dissociation constant, Kd(p140) was calculated from a reciprocal plot of nucleotide incorporation as a function of p55 concentration. WT and R182W p55 were shown to bind p140 with similarly high affinities (0.35nM for the WT and 0.11nM for the R182W p55)  . While t12-18 with varying dTTP concentrations. Rates of dTMP incorporation over time were used to calculate apparent Km(dTTP) and kcat values with the Michaelis-Menten steady-state kinetic model  of 26.6 ± 0.8 min-1 in the absence of p55 (kexo) was determined by plotting loss of substrate versus time over 5 minutes. In the presence of WT p55, p140’s kexo was 8.6 ± 2.3 min-1 while in the presence of R182W p55, p140’s kexo was 14.0 ± 1.2 min-1 . Pol γ holoenzymes were assembled in vitro with p140 and samples of each treated p55, and holoenzyme activity was evaluated by standard DNA synthesis assay. The half-life of WT p55 was determined to be 70.4 ± 4.4 min while R182W p55 was determined to be 1.0 ± 0.1 min •oligo(dT) 0.1 min . This 70POLG2 has been shown to be essential for life through a collection of studies that have revealed: (i) Homozygous knockout (POLG2-/-) mice die in utero at embryonic day E8.0–8.5, while heterozygous mice (POLG2+/-) are haplosufficient and develop normally [Drosophila melanogaster POLG2 cause lethality in the early pupal stage [POLG2 knockdown model [POLG2 were all heterozygous mutations [POLG2 mutation (R182W p55) was the first homozygous POLG2 mutation identified, and the affected infant presented with liver failure at 3 months of age and subsequently died at 9 months [normally . (ii) Nual stage  and  is detrimental to mitochondrial function. As expected, growth curves identified a growth defect for the patient fibroblasts. The doubling time was about twice as long as control cells. When we switched the fibroblasts to the galactose-based media significant cell killing was observed. Growth in galactose as the sole sugar source forces cells to produce energy via the OX-PHOS pathway thus ensuring we are examining mitochondrial energy production [As an initial screen, we assessed the growth rates of patient dermal fibroblasts grown in either glucose or galactose media to understand whether the mutation at Chr17: 62492543G>A in oduction –38. A suoduction  noted apoduction . Taken tPOLG and POLG2. Both mtDNA copy number and transcript levels were significantly reduced when grown in glucose.P55 functions to enhance p140’s replication of mtDNA, thus we determined the mtDNA copy number and mRNA transcript levels for The patient fibroblasts grown in glucose confirm that mitochondrial dysfunction is present and suggest R182W p55 may be the cause. However, unidentified mutations may also be present within the fibroblasts. To isolate the effect R182W p55 has on cellular function, we expressed R182W p55 in HEK293 cells as previously reported . These cin vitro. Double-stranded DNA binding affinity, p140 binding affinity, steady state stimulation of p140 DNA polymerase activity and stimulation of processive DNA synthesis by Pol γ were all similar for R182W p55 and WT p55. We found a reduction in the inhibition of exonuclease activity by R182W p55 as compared to WT p55. Together these results indicate that R182W p55 functions in a manner similar to WT p55 in vitro. However, during purification it was noted that the conditions had to be modified to obtain soluble protein. This suggested potential instability of the protein.The known biochemical functions of WT and R182W p55 were assessed POLG2 gene was found responsible for adult onset syndromic sensory neuropathy, ataxia and parkinsonism and mtDNA deletions [The R182W amino acid substitution lies at the base of the dimerization domain of p55 , and thieletions .POLG2, R182W. Our studies indicate that when R182W p55 is folded properly and in solution, it functions similarly to WT p55. However, R182W p55 has a significant stability defect that dramatically impairs its ability to function. This stability defect is likely caused by the loss of electrostatic interactions normally afforded by the Arg side chain. Ex vivo studies indicate that patient dermal fibroblasts have a growth defect, reduced mtDNA copy number and lower transcript levels for both p140 and p55. HEK293 cells expressing R182W p55 have across the board reduced OX-PHOS capabilities. Taken together the results strongly suggest defects in the R182W p55 protein are responsible for mtDNA depletion in the patient and the subsequent onset of symptoms [We have characterized the first clinically identified homozygous mutation of human


Inhibition of Aβ production by β-secretase (BACE) has been proposed as a potential therapeutic strategy for AD. However, BACE inhibitors lack specificity and have had limited clinical benefit. To better study the consequences of reducing BACE metabolism, specifically of APP, we used an antibody, 2B3, that binds to APP at the BACE cleavage site, inhibiting Aβ production. 2B3 was administered either directly into the lateral ventricles or by intraperitoneal injection to (platelet-derived growth factor promoter hAPP717V (PDAPP) mice and WT mice. 2B3 reduced soluble Aβ40 and βCTF  and improved memory for object-in-place associations and working memory in a foraging task in PDAPP mice. 2B3 also normalized the phosphorylation of the N-methyl-D-aspartate receptor NR2B subunit and subsequent extracellular signal–regulated kinase signaling. The importance of this NR2B pathway for OiP memory was confirmed by administering the NR2B antagonist, Ro25-6981, to 18-month-old WT. In contrast, 2B3 impaired associative recognition memory in young WT mice. These data provide novel insights into the mechanism by which selective modulation of APP metabolism by BACE influences synaptic and cognitive processes in both normal mice and aged APP transgenic mice. •Inhibition of APP metabolism by ICV 2B3 reduced Aβ and βCTF production•2B3 prevented the onset of memory impairments in PDAPP mice•2B3 restored NR2B receptor phosphorylation and subsequent ERK activity•NMDA-NR2B receptor mechanisms contributed to object-in-place memory•Directly inhibiting APP-BACE metabolism provides a therapeutic strategy for AD Aβ is pOne approach to altering APP metabolism is through the use of BACE1 inhibitors . HoweverIn the present study, we used a monoclonal antibody, 2B3, directed against the BACE1 cleavage site of APP to reduce BACE1 cleavage without influencing other BACE1 substrates . We hypo22.1Male PDAPP mice , and theAPP V717F transgene DNA. PDAPP-specific primers forward: 5’-ATCTGGCCCTGGGGAAAAAAG-3’ and reverse: 5’-GATGTCCTTCCTCCTCTGTTC-3’  amplified the hAPP V717F mutation. Control primers for MusA-Actin forward: 5’-CACCACACCTTCTACAATGAGCTG-3’ and reverse: 5’-TCATCAGGTAGTCAGTGAGGTCGC-3’ (Eurofins) targeted MusA-Actin.Before assignment to groups, the mice were genotyped according to protocols described previously . Brieflyn's were, therefore, 8 WT control and 7 WT 2B3.Experiment 1 established the age of onset of cognitive impairment, and experiment 2 assessed the effects of ICV infusion of 2B3 on cognition in the same animals. ICV administration was considered the most appropriate means to ensure the delivery of the antibody in the brain and thus provide a test of its putative actions in vivo as evidenced from our previous cell culture experiments. Experiments 1 and 2a consisted of 2 replications: The first replication comprised PDAPP (n = 14) and WT littermates (n = 15), which underwent a battery of object memory tests at 6-8, 10-12, and 14-16 months of age. Subsequently, these mice received ICV 2B3  or vehicIn experiment 3, PDAPP (n = 19) and WT littermate controls (n = 20) were used to assess the protective effects of long-term administration of 2B3 that was initiated before the onset of cognitive impairment. As long-term administration was not feasible using an ICV minipump delivery method, 2B3 was administered to PDAPP mice (n = 9) by once weekly IP injection at 20 mg/kg for a period of 15 weeks from 11 months of age. Vehicle was administered at an equal volume in control WT (n = 10) and PDAPP mice (n = 10). An additional WT untreated group (n = 10) was included to assess the potential impact of IP treatment on performance. The initial behavioral tests were carried out at 11 months of age, before 2B3 administration. All treatment groups were then subsequently matched in terms of their object contact times and discrimination ratios (data not shown). After a further 15 weeks of IP injections, memory function was tested at 15 months of age. These mice were tested on object memory tasks and a spatial working memory foraging task  to estabExperiment 4 evaluated the hypothesis that NMDA-NR2B receptor function was necessary for associative recognition memory in WT mice. A naïve cohort of 18-month-old WT littermate controls (n = 10) was administered the NR2B receptor antagonist Ro25-6981 or vehicle 30 minutes before behavioral testing. The experiment was run as a within-subject Latin-square design. A Latin-square is a commonly used experimental design to assess the effects of drug(s) and vehicle in the same animals on performance in a counterbalanced fashion.Mice were given a 48-hour rest interval between each injection as a washout period before the next phase of testing. Novel objects were used in each phase of drug testing, and their order was counterbalanced across animals.2.2In experiments 1 and 2, mice received 2 different object-based memory tasks; a novel object recognition task and a visuospatial OiP task  Fig. 2A.Figure 22.2.1To assess object novelty versus familiarity discriminations, 2 identical objects were placed in the center of the arena and mice were allowed to explore the objects for 10 minutes during the sample phase. Mice were given a total of 3 sample phases, each separated by a 5-minute retention interval, during which the mouse was returned to its home cage. Following the third sample phase, mice received either a 5-minute or 24 hour delay period, the order of which was counterbalanced within groups. During the test phase, 1 familiar and 1 novel object were then placed into the arena in identical spatial locations as the sample objects A. The mo2.2.2Four different objects were placed in the center of the arena in a square formation. Each object was approximately 15 cm from the walls and 25 cm apart from each other. Mice were placed in the center of the arena and exposed to the 4 different objects for 3 sample phases and a test phase as described for the object novelty test. In the test phase, conducted 5 minutes after the sample phase, the spatial location of 2 objects that were positioned diagonally opposite each other was switched B. The sp2.2.3For both recognition tasks, the animals' exploratory behavior was assessed during both the sample and test phases. Time spent exploring the objects was recorded in each phase. Object exploration was defined according to the methods described previously by 2.2.420) in an open arena with 1-cm-deep sawdust covering the arena floor. Mice were placed in the center of the arena and given 3 minutes to forage from each pot. Mice were removed once the liquid reward was consumed from each pot or the 3-minute limit was reached. Mice were given 1 training session/day. On each day, the location of the pots was moved to a new location to prevent the development of any systematic search bias in the test phase.To establish the generality of the cognitive changes promoted by 2B3 administration, mice that received longitudinal IP 2B3 injections were also assessed on a foraging-based spatial working memory task sensitive to age-dependent changes in PDAPP mice . BrieflyMice were then tested over the next 4 consecutive days with 1 session per day. During these sessions, the arena was set up with 6 pots arranged in a circular pattern, each 20 cm apart. The mouse was placed in the center of the arena and allowed to forage pots until they had consumed all 6 rewards or until a 10-minute period had elapsed from when the first pot was foraged. Following the trial, mice were returned to their home cage. The pots were then wiped clean with 70% ethanol wipes, and the milk solution replenished before the next mouse was tested. All test sessions were recorded onto a DVD player using an overhead camera.Foraging behavior was defined as a mouse jumping onto the rim of a pot and directing its nose in toward the bottom to consume a reward. An error was defined as a mouse returning to forage in a pot that had previously been foraged. A full description of errors can be found in 2.3Full details of the immunization protocol, hybridoma development, antibody characterization, production, and purification are provided elsewhere .In Experiments 2a and 2b, purified and sterilized 2B3 (1.2 mg/mL), control monoclonal IgG1κ antibody (experiment 2b) and vehicle (experiment 2a) were administered intracerebroventricularly via a 28G cannula surgically implanted into the left lateral ventricle . The minipumps were attached to the infusion cannula via a catheter and implanted subcutaneously between the scapulae. Minipumps were filled with 200 μL of antibody or vehicle . In experiment 2a, the vehicle was sterile PBS and was administered to both PDAPP mice (n = 11) and WT littermate control groups (n = 10). In experiment 2b, the WT control group received ICV infusion of a monoclonal IgG1κ.In experiment 3, mice received a weekly IP injection of 2B3 for 15 weeks. 2B3 was produced by ascites , before being purified and sterilized as described previously. PDAPP mice were administered 2B3 (IP) at a dose of 20 mg/kg at an average concentration of 3 mg/mL. Sterile PBS was administered at the same volume to vehicle PDAPP and WT mice control groups. After the completion of behavioral testing and 3 days after the final IP injection of 2B3, brain tissue was collected from all mice for biochemical analyses.2.42 mix, and a small hole was drilled through the skull of the animal 0.5 mm posterior and 1.2 mm lateral to Bregma. A 28G cannula was then inserted 3.0 mm ventral to the skull surface and fixed in place by dental acrylic. The minipump was carefully inserted into a subcutaneous pocket between the scapulae, the skin incision sutured, and the mouse allowed to recover in an incubator until independently mobile. Following standard postoperative care procedures, the mice were then housed individually for the duration of the study.During stereotaxic surgery, the mouse was anesthetized using an isoflurane/OPostoperative behavioral testing on the OiP associative recognition task occurred 9 days after surgery and used the procedure described in experiments 1 and 2. On day 14, following behavioral testing, the animals were culled and brain tissue collected for analyses.2.5Ro25-6981  was dissolved in sterile saline at a final concentration of 5 mg/mL. Mice were administered a single 10 mg/kg dose of Ro25-6981 or vehicle IP, 30 minutes before the start of the associative recognition OiP task. This dose was selected based on previously published behavioral work e.g., .2.6The hippocampus and cortex were dissected, snap-frozen in liquid nitrogen, and stored at −80 °C. Soluble proteins were extracted from brain samples as previously described . For the2.7ELISAs for the quantification of Aβ were carried out as previously described , or as r2.8p’s > 0.5 (data not shown). ELISA data were analyzed using either Student’s t-test or a Kruskal-Wallis test with Dunn’s test along with a Bonferroni correction for multiple comparisons. All data were subject to Levene’s and Shapiro-Wilks’ tests for data normality before analysis. Appropriate transformations were carried out when necessary.All statistical analyses were performed using IBM SPSS statistics. The behavioral data conformed to the assumptions of analysis of variance (ANOVA) and were analyzed using a mixed measures design. Significant interactions were assessed using tests for simple main effects. Western blot data were analyzed using one-way ANOVA followed by Tukey's post hoc analysis. WT data were collapsed across treatment (untreated and vehicle) for the analysis of the cognitive and behavioral effects of 2B3 administration as no group differences were observed between WT groups www.imagej.nih.gov/).Data generated from ELISA assays were quantified by comparing data to standard curves from each plate using GraphPad Prism 4 and normalized to total protein concentration. One PDAPP 2B3-treated mouse was found to be an extreme outlier across the ELISA analyses of the 2B3 ex vivo tissue, as determined by SPSS Tukey box plots and more conservative methods for labeling outliers, as described previously . No tran3Experiment 1 used object novelty and OiP tasks to determine the nature and age of onset of memory deficits in PDAPP mice. In the second stage of the study, experiment 2a, the same animals were administered the APP antibody 2B3, at 17-18 months of age, via osmotic minipumps for a period of 14 days. Having established that 2B3 reversed pre-existing associative recognition memory impairment, experiment 3 determined whether longitudinal peripheral administration of 2B3 prevented the onset of cognitive decline in PDAPP mice.3.1p’s < 0.01 . There were no significant interactions involving genotype and delay for either task  = 2.5, p > 0.1, and no genotype × age interaction, F = 2.8, p > 0.1. In contrast, PDAPP mice were significantly impaired relative to control mice only at 14-16 months of age for the OiP task  = 9.2, p < 0.05. Subsequent tests of simple main effects revealed that PDAPP mice showed a significant main effect of age with a reduction in OiP memory performance across age ranges, F = 9.8, p < 0.001. Further analysis revealed that PDAPP mice showed a significant memory deficit compared with WT mice only at 14-16 months of age, F = 49.9, p < 0.001. There was no significant interaction involving delay, F < 1. The analysis of DR values confirmed that PDAPP mice showed an age-dependent deficit in associative recognition memory, without affecting object novelty/familiarity discriminations.An analysis of DRs similarly showed an identical pattern with PDAPP mice performance, as well as WT mice, on the novelty test at all ages as indicated by no main effect of genotype, task see A and 3B.2(3) = 2.7, p > 0.1) and a significant, X2(3) = 10.5, p < 0.05, 32-fold increase in soluble Aβ42 levels by 15 months of age  = 3.27, p < 0.05. Tests of simple main effects revealed that PDAPP mice administered ICV 2B3 showed a significant improvement in performance compared to both their pre-administration, p < 0.01, and to vehicle PDAPP mice performance, p < 0.001. In contrast, vehicle PDAPP mice remained significantly impaired relative to WT controls, p < 0.001, and 2B3 PDAPP mice were not significantly different to WT mice, p > 0.5  = 3.41, p < 0.05. Testing for simple main effects further revealed that, although vehicle PDAPP mice showed a decline in OiP performance across pre- and post-treatment time points, p < 0.01, IP administration of 2B3 prevented this decline, p > 0.05. Moreover, both WT and 2B3 PDAPP mice showed comparable performance (p > 0.05) and both groups performed better than vehicle PDAPP mice, p values <0.001  = 0.16, p > 0.1, or treatment group × time interaction, F = 0.27, p > 0.1,  = 3.68, p < 0.05. Tests of simple main effects showed that 2B3 PDAPP mice made less errors than their pre-drug assessment at 11 months of age, p < 0.05 and less errors than vehicle PDAPP mice, p < 0.01  = 3.85, p < 0.05. Simple main effects analysis revealed that 2B3 PDAPP mice made fewer repeat errors than vehicle PDAPP mice, p < 0.05, and that there was no significant change in the number of repeat errors compared to pre-drug performance, p > 0.05. In contrast, vehicle PDAPP mice showed an age-dependent increase in the total number of repeat errors compared to pre-drug assessment, p < 0.05. Vehicle PDAPP mice also made more repeat errors than WT mice, p < 0.01. This impairment was not present in 2B3 PDAPP mice, p > 0.05.To establish the generality of the improvement in associative OiP memory with 2B3, PDAPP mice were also tested on a spatial working memory foraging task . Time top > 0.05 , which ip > 0.05 D and E. p < 0.01 D. In con < 0.001 D. WT micg memory E. An ANO3.4In experiment 2a, Western blot analysis showed that ICV 2B3 administration caused no change in total levels of BACE1, F = 1.95, p > 0.1, or presenilin 1 (PS1), F = 1.40, p > 0.1, in the hippocampus of PDAPP mice  = 2.30, p > 0.1, and PS1, F < 1  = 8.49, p < 0.01 in ICV 2B3 PDAPP mice  = 1.32, p > 0.1, or IP administration, t(17) = 1.45, p > 0.1  = 2.28, p < 0.05, and a (nonsignificant) 66% reduction in levels of soluble Aβ42, t(18) = 1.01, p > 0.1, in the hippocampus  = 2.22, pocampus . In experol mice . Endogenrol mice H-K.F = 2.72, p > 0.05]. A similar pattern was evident in experiment 3 after IP 2B3 administration  = 0.12, p > 0.1, respectively; F = 2.36, p > 0.05  = 3.86, p < 0.05, PDAPP vehicle mice showed reduced pY1472 compared with WT vehicle mice, p < 0.05  = 16.11, p < 0.001 and IP (experiment 3) studies, F = 5.30, p < 0.01. Post hoc Tukey's analyses revealed a reduction in vehicle PDAPP mice compared with WT mice in both ICV and IP groups . Neither ICV nor IP 2B3 PDAPP mice differed relative to their respective WT control mice, p values > 0.1.In experiment 2a, there were no significant differences in the total expression levels of the synaptic marker PSD95 or NMDA receptor subunits, NR1, or NR2B in hippocampal synaptosomes of ICV  in synaptosomes of ICV vehicle PDAPP compared with WT (p < 0.01) and ICV 2B3 PDAPP mice  = 7.03, p < 0.01, with a significant reduction compared with both WT (p < 0.05) and ICV 2B3 PDAPP mice (p < 0.01). There was no significant difference between WT and ICV 2B3 PDAPP mice, p > 0.05  is downstream of NMDA signaling and plays a role in regulating memory processes . There wp > 0.05 D.3.5p's > 0.05 (p < 0.001) and Ro-25-6981 (p < 0.01) mice explored objects in novel locations more than those in familiar locations  compared with direct ICV administration. Further research is required to assess the temporal dynamics of the interactions of 2B3 with APP processing in vivo. Nevertheless, our data present clear evidence that, even under a restricted set of conditions, 2B3 has an impact on APP processing and memory.Excess Aβ production leads to changes in the dynamic properties of hippocampal synaptic plasticity; promoting the induction of LTD and impairing the induction of LTP . This chExcess Aβ production can also result in activation of Fyn kinase, which leads to phosphorylation of NR2B at Y1472 and stabilizes expression of the receptor at the membrane surface. The effect of Aβ on Fyn kinase is counteracted by elevation of STEP ; for exaIt is important to acknowledge that other anti-APP-BACE1 and immunization approaches have been investigated. Thus, https://www.eisai.com; aducanumab, In terms of the clinical relevance of our findings, evidence is beginning to emerge that some antibody-based therapies have a positive impact on amyloid pathology and may be disease-modifying in amyloid-positive patients (e.g., BAM2401, As highlighted by In conclusion, the present study has shown that selectively influencing APP metabolism by reducing BACE1 activity by steric hindrance with an antibody, reversed and protected against an age-dependent associative recognition memory deficit in PDAPP mice. ICV administration of 2B3 reduced levels of soluble Aβ and βCTF without affecting total levels of APP, and both ICV and chronic IP administration normalized the phosphorylation of NMDA-NR2B receptors. These novel findings provide important evidence that selective inhibition of APP processing at the BACE1 cleavage site can improve both memory and markers of synaptic pathology in a mouse model of age-related amyloid accumulation. Finally, the results contribute to other findings suggesting that modification of APP processing, and associated downstream glutamatergic signaling cascades, may be beneficial in populations with altered APP metabolism, including aged individuals  and thosAll authors declare that they have no competing financial interests.


The engineering of metal binding into a cellulase increases its temperature stability while maintaining its other catalytic properties. 2+ site, the thermal tolerance was increased by more than 10°C and was paralleled by an increase in the catalytic optimum temperature by more than 5°C.The development of robust enzymes, in particular cellulases, is a key step in the success of biological routes to ‘second-generation’ biofuels. The typical sources of the enzymes used to degrade biomass include mesophilic and thermophilic organisms. The endoglucanase J30 from glycoside hydrolase family 9 was originally identified through metagenomic analyses of compost-derived bacterial consortia. These studies, which were tailored to favor growth on targeted feedstocks, have already been shown to identify cellulases with considerable thermal tolerance. The amino-acid sequence of J30 shows comparably low identity to those of previously analyzed enzymes. As an enzyme that combines a well measurable activity with a relatively low optimal temperature (50°C) and a modest thermal tolerance, it offers the potential for structural optimization aimed at increased stability. Here, the crystal structure of wild-type J30 is presented along with that of a designed triple-mutant variant with improved characteristics for industrial applications. Through the introduction of a structural Zn This classification facilitates the grouping of GHs according to structural, mechanistic and evolutionary aspects independent of substrate specificities.Concerns about climate, increasing energy demands and the limited long-term supply of fossil fuels, as well as ethical considerations, have promoted research on more sustainable lignocellulosic ‘second-generation’ biofuels. They rely on the deconstruction of cellulose and hemicellulose from feedstocks such as switchgrass into monosaccharides. One step in this process, saccharification, employs mixtures of thermally and chemically robust glycoside hydrolases (GHs). These mixtures typically make use of the combined activities of endoglucanases (EC 3.2.1.4), β-glucosidases (EC 3.2.1.21) and cellobiohydrolases . The Carbohydrate-Active enZymes database , which produces cellobiose as its primary product, was identified and analyzed from metagenomic data originating from a switchgrass-adapted microbial community  within the Ig-like domain. However, the more recent dbCAN2 meta server (http://cys.bios.niu.edu/dbCAN2) does not identify a CBM. With 54% sequence identity, an uncharacterized family 9 GH from T. composti is the closest homolog of J30. Of those with solved structures, the leaf-branch compost CelG  with 32 and 31% identity, respectively. Among the less similar family members found in the Protein Data Bank  and a glucosaminidase from Photobacterium profundum . The crystal structure of the triple mutant showed the successful introduction of a Zn2+ ion at the intended binding site. Our circular-dichroism (CD) and differential scanning fluorimetry (DSF) data demonstrate a successful increase in the melting temperature of J30. An enzymatic analysis of the enzyme variants showed an increase in the catalytic optimum temperature by 5–10°C. Our data support the proposed role of structural metal ions in promoting thermal stability .Escherichia coli NEB Express cells grown in TB medium with 50 µg ml−1 kanamycin and 2 mM MgSO4 at 200 rev min−1. The bacterial suspensions were incubated at 37°C before induction with 0.5 mM IPTG at an OD600 nm of 4 (J30 wt) or 1 (J30 CCH) and at 20°C thereafter. The protein expressions lasted 17 h (J30 wt) or 19 h (J30 CCH) until an OD600 nm of 20 (J30 wt) or 20.5 (J30 CCH) was attained.Wild-type J30 (J30 wt) and J30 CCH, each carrying a C-terminal polyhistidine tag, were produced in M NaCl, 25 mM HEPES pH 7.4 with 1 mM PMSF using an Avestin EmulsiFlex-C3 homogenizer. The proteins were purified using an ÄKTAexplorer 100 Air by nickel-affinity (5 ml HisTrap HP or FF) and size-exclusion chromatography . All of the media and buffers used to produce and isolate J30 CCH also contained 10 µM ZnCl2.The cells were lysed in a buffer consisting of 0.15 2.2.et al., 2017−1 purified enzyme, the protein was crystallized in 0.1 M trisodium citrate dihydrate pH 5, 15%(w/v) 2-propanol, 10%(w/v) PEG 10 000. Suitably sized crystals were obtained after 3 d of growth at room temperature using the sitting-drop vapor-diffusion method. J30 CCH crystals were prepared in the same manner. All drops consisted of 1 µl protein solution and 0.5 µl reservoir solution.Initial sparse-matrix crystallization screening of wild-type J30 was conducted using the following screens: Berkeley  glycerol and flash-cooled in liquid nitrogen. Native J30 data sets were collected on beamline 7-1 at the Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory, and the J30 CCH diffraction data were recorded on the Berkeley Center for Structural Biology beamline 5.0.1 of the Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory. All data were measured using an oscillation angle Δφ of 1° and ADSC Quantum 315r detectors.The crystals were transferred through 20% served as a search model for J30 wt (top TFZ score of 19.7), which in turn was used to generate the J30 CCH maps. Model improvement employed alternating rounds of manual modelling using Coot .All data sets were processed using the 2.4.−1 enzyme, 150 mM NaCl, 25 mM HEPES pH 7.4, 10 µM ZnCl2 and SYPRO Orange Protein Gel Stain  at 5×. Fluorescence signals were recorded from 25 to 99°C.DSF assays comparing the thermal stability of J30 wt and J30 CCH were performed on an Applied Biosciences StepOnePlus Real-Time PCR System in triplicate on a 30 µl scale containing 0.5 mg ml2+-titration samples were prepared by a 1 h incubation of J30 CCH with a 150-fold stoichiometric excess of EDTA pH 7.4 and dialysis for 18 h against 0.15 M NaCl, 25 mM HEPES buffer pH 7.4. A 1 mg ml−1 enzyme solution was incubated with ZnCl2 at various concentrations for 30 min before the addition of the dye. The DSF assays were carried out as above and at final concentrations of 0.5 mg ml−1 J30 CCH, 0–10 µM ZnCl2, 0.15 M NaCl, 25 mM HEPES pH 7.4 with SYPRO Orange Protein Gel Stain at 5×.The Zn2.5.E. coli were recorded from 20 to 85°C with a Jasco J-815 CD spectrometer using 1 mm cuvettes. The assay concentrations were 0.1 mg ml−1 enzyme, 150 mM NaCl, 25 mM HEPES pH 7.4, 10 µM ZnCl2. Three accumulations were recorded in the wavelength range 200–250 nm applying 0.2 nm data pitches.Melting curves of J30 wt and J30 CCH from 2.6.−1 enzyme, 0.1 M MES pH 6, 10 µM ZnCl2, and 0.25 mM 4-nitrophenyl β-d-glucopyranoside , 4-nitrophenyl β-d-cellobioside  or 4-nitrophenyl β-d-cellotrioside . Positive controls contained a final concentration of 3.6 M NaOH instead of enzyme. Negative controls contained buffer instead of enzyme, and were subtracted before internal standardization against the alkaline hydrolysis. Triplicate samples were incubated at 50°C in Applied Biosciences Veriti 96-Well Thermal Cyclers, cooled to 4°C, transferred to 96-well clear flat-bottom plates and mixed with 2%(w/v) sodium carbonate in a 1:1 volume ratio. The absorbance at an incident wavelength of 405 nm was measured on a Molecular Devices SpectraMax M2.Reactions were set up on a 50 µl scale using 15 µg ml2.7.−1 enzyme, 0.3 mMpNPC , 10 µM ZnCl2, 31.3 mM citrate and, depending on the pH, 4.7–51.1 mM lactate and 28.4–59.4 mM phosphate. 100 µl reactions were set up in triplicate in 96-well plates on BioExpress GeneMate IsoFreeze PCR racks and incubated for 30 min in Applied Biosciences Veriti 96-Well Thermal Cyclers. The sample-cooling and transfer, reaction-quenching and readout steps were carried out as described above, except that Beckman Coulter Biomek FX and NXP robots were used for all of the liquid handling in the 96-well format plates. Negative controls contained buffer instead of enzyme and were subtracted.The assay conditions comprised 15 µg ml3.3.1.The crystal structure of J30 was solved to a resolution of 1.7 Å by molecular replacement Table 1. The 64 C. thermocellum , have additional upstream amino acids that contribute to an eighth β-strand at the interface to the catalytic domain  and four  to form two strictly antiparallel β-sheets. The latter sheet is bifurcated owing to Pro74 kinking β-strand 7 into two halves. The N-terminus is located at the end of a cavity near the catalytic domain. In both crystal structures presented here a citrate ion occupies the space where the closest homologs of J30 within GH family 9, including CbhA from 6-barrel structure composed of 12 α-helices arranged in an alternating pattern as is typical within GH family 9 6-barrel, α-helix 17 is the only conserved helix.The predominant architectural element of the catalytic domain is an (α/α)The substrate-binding channel extends from a region near the N-terminal ends of the inner barrel helices (corresponding to where the nonreducing end of the bound substrate would be) towards a more unstructured portion of J30 flanked mostly by random coil Fig. 1. It runs3.2.2+ ion in the predicted site . Metal coordination is mediated by the side chains of Cys98, Cys114, His115 and His143, and effectively replaces one hydrogen bond formed by His115 and Tyr143 in the wild-type enzyme . Among the GH family 9 structures with a homologous Zn2+-binding site, CtCel9A, AaCel9A , have a serine taking the role of the histidine  or ring stacking (Trp408). The side chain of the latter residue is stabilized through a moderately conserved hydrogen bond to Tyr528. The hydrogen bond between the main-chain N atom of Gly304 and the carbohydrate moiety in the −1 position is only shared between J30 and its closest homologs LC-CelG, CtCel9A, AaCel9A and CtCbhA.The carbohydrate ring in the +1 position is stacked between the side chains of Trp226 and Tyr519. All GH9 family members with solved structures contain aromatic residues in these positions. This ring is further held in position through hydrogen bonds involving His467 and Arg469, both of which are highly conserved among the same structures except for the less closely related PpGlcNase and the chitobiase from 2+-introduction site from the active site is a result of the side chain of His143 pointing in the opposite direction to Asp144 and Asp147. As this orientation of the His143 side chain is similar to that of Tyr143 in J30 wt, a negative impact of metal ion binding on the catalytic activity of the enzyme would not be expected. In contrast, stabilization of the stretch of random coil containing His143, Asp144 and Asp147 could be explained by the triple mutation and introduction of Zn2+ through connection of the three loops containing Ala98, Gly114 and His115, and Tyr143 through the metal ion.Figs. 23.4.a). Based on the inflection points of the curves, the Tm of J30 CCH (70.1°C) is 11.2°C higher than that of the wild-type enzyme (58.9°C). That this increase is owing to the presence of the Zn2+ ion was confirmed by its removal using EDTA, which reduced the Tm of the triple mutant to the same level as that of J30 wt . Reintroduction of the ion into J30 CCH fully restores the thermal tolerance of the enzyme. CD melting curves support these observations . The local minima at 208 and 222 nm typical of α-helical content are consistent with the structure of the catalytic domain. Their decreases in amplitude with increasing temperatures probably reflect a loss of structural integrity of the catalytic domain. Based on the DSF data, the J30 CCH mutant has a modest 5 µM affinity for Zn2+  with substrate hydrolysis by 3.6 M NaOH (representing 100%; Fig. 5pNPG and produce more detectable pNP from pNPC than from the cellotetraose analog pNPG3. While the triple mutant produced slightly more pNP from pNPG3 than the wild-type enzyme at 50°C, both ultimately appear to favor the hydrolysis of pNPG3 to cellobiose and noncleavable pNPG over the production of cellotriose and pNP, thereby paralleling the experimental data for AaCel9A (Eckert et al., 2002J30 has been reported to hydrolyze the cellotriose analog 3.6.pNPC as a substrate (Fig. 62+ ion increases the temperature optimum of J30 for pNPC turnover by 5–10°C, and its influence on catalysis is merely physical and not chemical. These findings are confirmed by the DSF and CD data, and are consistent with prior characterization of the wild-type J30 enzyme (Gladden et al., 2014The rate of product formation in enzymatic catalysis, and in a hydrolysis reaction in particular, is inherently dependent on the temperature and pH. The nature of these relationships in the case of J30 wt and J30 CCH was studied using te Fig. 6. Both en4.2+ binding on the thermal tolerance of the cellulase CtCel9A has been shown previously, as has the correlation of a Zn2+-binding site with enzyme activity and structural integrity (Chauvaux et al., 19902+ ion and the side chains of Cys98, Cys114, His115 and His143 (J30 CCH; Fig. 2via the Zn2+ ion. The structural and biochemical data strongly suggest that the metal-binding site is purely structural, yet it is also proximal to the active-site residues Asp144 and Asp147 and the region of the substrate cleft where the reducing end binds.The positive effect of Ca2+ ions. Three Ca2+-binding sites are found in CtCel9A, two of which, in varying combinations, are found in AaCel9A, CtCbhA and LC-CelG. Two of the Ca2+ sites are found in loop regions with backbone geometries different to those in J30, corresponding to J30 residues 175–198 and 471–475 (Juy et al., 1992et al. (19952+ ion into J30 by point mutations in this region may work in a similar manner to the Zn2+ example, but would be likely to require loop remodeling in the case of the other two sites.In contrast to its closest homologs, J30 does not contain bound Ca al. 1995. In J30,2+-binding site seen in homologs of the target enzyme, and as a result have improved the thermal stability and the temperature optimum of the enzyme. This strategy is likely to be proven to be promising in the future in a more general context: the de novo introduction of metal ion binding into proteins. Such an approach is undoubtedly more challenging when there are no metal ion-binding homologs that can be used as a reference. In particular, recent efforts show how interaction networks beyond the geometry of the immediate site to be designed need to be considered (Guffy et al., 2016et al., 1999et al., 2016et al., 2011Unlike the incorporation of disulfide bonds to cross-link otherwise more loosely connected sections of a polypeptide chain, our approach is not limited to secreted proteins. We have taken advantage of a Znwild-type J30, 5u0hPDB reference: J30 CCH, 5u2oPDB reference: 10.1107/S2059798318006678/yt5103sup1.pdfSupplementary Figure S1.. DOI:


It has been observed that converting stevioside and rebaudioside A to their corresponding ketones by switching the doubly bonded methylene on C-17 for a ketone group actually removes the sweet taste properties of these molecules completely. Regenerating the original molecules tends to restore the sweet taste of both the steviol glycosides. Thus this C16-C17 methylene double bond in rebaudioside A and stevioside can be regarded as a pharmacophore essential for the sweetness property of these molecules.The structure activity relationship between the C Stevia rebaudiana. Steviol  and stevioside  are swee Steviol  is the aet al. in 1991 (methyl)triphenyl phosphonium iodide. However there are no other similar literature reports available for similar synthetic transformations on reb A or stevioside molecules.Preparation of steviol ketone has previously been reported in the literature . This PhWe first repeated the literature reported Wittig methylenation reaction procedure on steviol acetate ketone to see if it worked in our hands, but we were not very successful with this procedure. Further Wittig reaction was also tried on reb A peracetate nor-ketone and stevioside peracetate nor-ketone. The reaction conditions seemed to deacetylate the starting molecules and the resulting reaction mixtures seemed to be mixtures of several products. Apparently this reaction did not seem to work for any of these molecules.17 methylene form using TiCl4-Zn-CH2Br2 reagent −, Calcd. 317.44 for C20H30O3[M−H]−; HPLC .l Method : SteviosSteviol (1) - (ii) Enzymatic Method +, Calcd. 383.48 for C22H32O4[M+Na] +; HPLC .Steviol Acetate Ketone (3) +, Calcd. 385.45 for C21H30O5 [M+Na]+, HPLC.e Ketone  [3]. SteMethylenation of Steviol Acetate Ketone +, Calcd. 383.48 for C22H32O4[M+Na]+, HPLC .e Ketone . SteviolSteviol nor-ketone (4). Steviol acetate nor-ketone  was refluxed in 2 M KOH (20 mL) for one hour and then cooled. The cooled reaction mixture was then acidified with 30% HCl to pH = 2.5 whereupon the solution becomes milky and a white precipitate appears. It was extracted with ethyl acetate (3 × 15 mL), dried over sodium sulfate and then evaporated on a rotavapor at 30 °C under reduced pressure, yield = 30 mg, 85%. TLC : Rf = 0.28; 1H-NMR (CDCl3) δ = 1.205 , 0.940 ; 13C-NMR: δ = 214.201 ; ESI-MS m/z: 319.4 [M−H]−, Calcd. 319.42 for C19H28O4[M−H]−, HPLC . A similar procedure was followed for de-acetylation of 2 to 1. Steviosideperacetate (5). In a 500 mL round bottom flask, stevioside  was dried overnight in a vacuum oven at 75 °C. To this was added dry pyridine (200 mL) followed by cooling to 0 °C and then addition of acetic anhydride (350 mL). It was allowed to warm up to room temperature and then stirred at room temperature for 48 h. The reaction was then poured on a beaker containing ice and shaken vigorously. It was then extracted with diethyl ether (3 × 50 mL) followed by washing the organic layer with cold water (3 × 30 mL), 5% hydrochloric acid (3 × 30 mL), cold saturated sodium bicarbonate till no further evolution of CO2 was seen (3 × 40 mL) and then finally by brine (30 mL). The organic layer was dried over sodium sulfate and then evaporated to get the crude per-acetylated stevioside which was purified by silica gel flash chromatography (230–400 mesh) using a stepwise gradient of dichloromethane-methanol from pure dichloromethane up to 10% methanol in dichloromethane, yield = 10.00 g (77%). TLC : Rf = 0.52; 1H-NMR (DMSO) δ = 6.016 ; 13C NMR δ = 153.016 , 103.792 ; ESI-MS m/z: 1289.8 [M+Na]+, Calcd. 1290.27 for C60H82O29 [M+Na]+.Stevioside per acetate Ketone (7). Stevioside per-acetate  was taken up in a mixture of THF (4.0 ml) and water (3.5 mL). To this was added freshly made 4% aqueous solution of OsO4 (0.5 mL). After 10 min the reaction mixture had become deep brown and to this NaIO4 (840 mg) was added slowly over a period of 2–3 min and the mixture was left for stirring at room temperature for 17 h. It had become a thick white suspension by now and was extracted with ethyl acetate (2 × 40 mL). The organic layer was dried over sodium sulfate and evaporated on arotavapor at 30 °C under reduced pressure to give the crude stevioside per-acetate ketone, which was chromatographed on silica gel 60 (70–230 mesh) with a stepwise gradient from pure dichloromethane to 2.5% methanol in dichloromethane. Pure ketone started coming out in fractions collected at 2% dichloromethane in methanol, yield = 378 mg (27%). TLC : Rf = 0.42; 1H-NMR (DMSO) δ = 6.024 ; 13C-NMR δ = 191.228 , peaks at δ 153.016 & 103.792 present in 5 disappear here; ESI-MS m/z: 1292.0 [M+Na]+, Calcd. 1292.24 for C59H80O30 [M+Na] +.Methylenation of stevioside acetate ketone and re-generation of stevioside. Stevioside peracetate ketone  was taken up in anhydrous dichloromethane (10.5 mL) and to this solution was slowly added, under nitrogen, Zn/CH2Br2/TiCl4 reagent . The reaction mixture was stirred for 18 h. at room temperature when the TLC in DCM–MeOH (20:1) showed disappearance of ketone. The reaction was further monitored by HPLC on an amino column. On the basis of HPLC and LC-MS of t = 0 and t = 18 h. samples, the reaction appeared complete. It was then quenched by pouring into a biphasic mixture of saturated sodium bicarbonate (20 mL) and ethyl acetate (10 mL). The aqueous layer was extracted two more times with ethyl acetate (10 mL ea.). The combined ethyl acetate layer was dried over sodium sulfate and evaporated on a rotavapor at 30 °C under reduced pressure to afford a crude product that was taken up in anhydrous methanol (10 mL). To this was added anhydrous sodium methoxide (200 mg). It was stirred at room temperature for 2 h and then neutralized by 0.5% HCl to pH ~ 6.5–7.0. The methanol was removed on a rotavapor under reduced pressure. A 1.0 mL size short column of reverse phase silica, C18, was packed in 50% acetonitrile-water and then washed with 50–100 mL double distilled water. The de-acetylated product was dissolved in minimum amount of water and loaded on this C18 column followed by washing with water (100 mL). Elution with 90% acetonitrile-water (50 mL) resulted in pure stevioside, as shown by HPLC . The acetonitrile solution was evaporated and dried to get pure stevioside, yield = 208 mg (68%).Stevioside ketone (9). Stevioside peracetate ketone  was taken up in anhydrous methanol (10 mL). To this was added anhydrous sodium methoxide (200 mg). It was stirred at room temperature for 2 h. and then neutralized by 0.5% HCl to pH ~ 6.5–7.0. The methanol was removed on a rotavapor under reduced pressure followed by desalting on a pre-packed C-18 cartridge (500 mg) from Varian. The de-acetylated product was further purified via preparative HPLC on a C-18 column to get pure stevioside ketone, yield = 158 mg (55%). 1H-NMR (DMSO) δ = 1.212 , 0.983 ; 13C-NMR: δ = 191.230 ; ESI-MS m/z: 809.1 [M+H]+, Calcd. 807.85 for C37H58O19 [M+H] +.Per-acetylated reb A (6). In a 500 mL round bottom flask, rebaudioside A  was dried overnight in a vacuum oven at 75 οC. To this was added dry pyridine (150 mL) followed by cooling it to 0 οC and then addition of acetic anhydride (250 mL). It was allowed to warm up to room temperature and then stirred for 48 h. The reaction was then poured on a beaker containing ice and shaken vigorously. It was then extracted with diethyl ether (3 × 50 mL) followed by washing the organic layer with cold water (3 × 30 mL), 5% hydrochloric acid (3 × 30 mL), cold saturated sodium bicarbonate till no further evolution of CO2 was seen (3 × 40 mL) and then finally by brine (30 mL). The organic layer was dried over sodium sulfate and then evaporated to get the per-acetylated rebaudioside A as a single spot on TLC, yield = 6.5 g (80%). TLC : Rf = 0.36; 1H-NMR (DMSO) δ = 5.982 ; 13C-NMR δ = 152.890 , 104.292 ; ESI-MS m/z: 1578.5 [M+Na]+, Calcd. 1578.52 for C72H98O37[M+Na]+.Rebaudioside A Per-Acetate Ketone (8). Rebaudioside A peracetate  was taken up in a mixture of THF (4.0 mL) and water (3.5 mL). To this was added freshly made 4% aqueous solution of OsO4 (0.5 mL). After 15 minthe reaction mixture had become deep brown and to this NaIO4 (840 mg) was added slowly over a period of 2–3 min and the mixture was left for stirring at room temperature for 18 h. It had become a thick white suspension by now and was extracted with ethyl acetate (2 × 40 mL). Organic layer was dried over sodium sulfate and evaporated on a rotavapor at 30 °C under reduced pressure to get crude rebaudioside A per-acetate ketone that was chromatographed on silica gel 60 (70–230 mesh) with a slow stepwise gradient from pure dichloromethane to 2.5% methanol in dichloromethane, yield = 220 mg (30%). TLC : Rf = 0.28; 1H-NMR (DMSO) δ = 5.982 , 13C-NMR δ = 215.749 , peaks at δ152.890 and 104.292 present in 6 disappear here; ESI-MS m/z: 1580.0 [M+Na]+, Calcd. 1580.5 for C71H96O38[M+Na]+.Methylenation of Rebaudioside A Acetate Ketone and re-generation of reb A. Rebaudioside A per- acetate ketone  was taken up in anhydrous dichloromethane (4 Ml). To this solution was slowly added under nitrogen, Zn/CH2Br2/TiCl4 reagent . The reaction mixture was stirred at room temperature and was monitored by HPLC on an amino column . A series of aliquots of the reaction mixture were taken out at t = 18, t = 41 and t = 72 h  to monitor the reaction. They were first quenched with sat. sodium bicarbonate and ethyl acetate (1:1) followed by isolation, drying and evaporation of ethyl acetate layer and then de-acetylation. On the basis of HPLC and LC-MS of t = 0 and t = 18, 41, 72 h. samples the reaction seemed complete after 72 h. The reaction was then quenched by pouring into a biphasic mixture of saturated sodium bicarbonate (10 mL) and ethyl acetate (5 mL). The aqueous layer was extracted two more times with ethyl acetate (5 mL ea.). The combined ethyl acetate layer was dried over sodium sulfate and evaporated on a rotavapor at 30 οC under reduced pressure to get a crude product which was only 37 mg. The crude was taken up in anhydrous methanol (4 mL). To this was added anhydrous sodium methoxide (19 mg). It was stirred at room temperature for 2 h. and then neutralized by 0.5% HCl to pH ~ 6.5–7.0. The methanol was removed on a rotavapor under reduced pressure. A 500 mg pre-packed C-18 cartridge from Varian was first treated with 50% acetonitrile-water and then washed with 15–20 mL double distilled water. The de-acetylated product was dissolved in minimum amount of water and loaded on C-18 column followed by washing with water (15 mL). Elution with 90% acetonitrile-water (15 mL) resulted in pure reb A as shown by HPLC. The acetonitrile solution was evaporated and dried to get pure reb A, yield = 17 mg (14.5% yield).Reb A ketone (10). Rebaudioside A peracetate ketone  was taken up in anhydrous methanol (4 mL). To this was added anhydrous sodium methoxide (19 mg). It was stirred at room temperature for 2 h. and then neutralized by 0.5% HCl to pH ~ 6.5–7.0. The methanol was removed on a rotavapor under reduced pressure followed by desalting on a pre-packed C-18 cartridge (500 mg) from Varian. The de-acetylated product was further purified via preparative HPLC on a C-18 column to get pure reb A ketone, yield = 31 mg (22%). 1H-NMR (DMSO) δ = 1.204 , 0.979 ; 13C-NMR: δ = 215.457 ; ESI-MS m/z: 992.1 [M+Na]+, Calcd. 991.98 for C43H68O24[M+Na]+.17 with a ketone actually removes the sweet taste properties of these molecules completely. Upon regenerating the original molecules, the sweet taste of both the steviol glycosides were found to be restored. These findings indicate that the C16-C17 part of reb A and stevioside is an essential pharmacophore for the sweetness properties of these molecules. This is also the first literature report of stevioside and reb A peracetate and its novel ketones derivatives. The methylenation reaction of these ketone derivatives using TiCl4-Zn-CH2Br2 reagent is also the first such report in the literature.We have investigated the relationship between the presence or absence of the methylene double bond on the aglycone of steviol glycosides and the corresponding impact on taste. We have found that converting stevioside and reb A to their corresponding ketones by switching the doubly bonded methylene on the C


Increasing radiotherapy total dose in the whole tumour volume has been shown to be deleterious. Functional imaging with positron emission tomography (PET/CT) offers the potential to identify smaller and biologically meaningful target volumes that could be irradiated with larger doses without compromising Organs At Risk (OAR) tolerance. This study investigated four scenarios, based on 18FDG- and 18F-miso PET/CT before the start of radiotherapy and 18FDG PET/CT during the radiotherapy (42Gy). The plans were based planned on a standard plan delivering 66 Gy (plan 1) and on three different boost strategies to deliver 74Gy total dose in pre-treatment 18FDG hotspot (70% of SUVmax) (plan 2), pre-treatment 18F-miso target (SUVmax > 1.4) (plan 3) and per-treatment 18FDG residual (40% of SUVmax). (plan 4).Twenty-one NSCLC patients, selected from a prospective phase II trial, had 18FDG hotspot, 38.9 cc (± 14.5) for 18F-miso and 36.0 cc (± 10.1) for per-treatment 18FDG. In standard plan (66 Gy), the mean dose covering 95% of the PTV (D95%) were 66.5 (± 0.33), 66.1 (± 0.32) and 66.1 (± 0.32) Gy for 18FDG hotspot, 18F-miso and per-treatment 18FDG. In scenario 2, the mean D95% was 72.5 (± 0.25) Gy in 18FDG hotspot versus 67.9 (± 0.49) and 67.9 Gy (± 0.52) in 18F-miso and per-treatment 18FDG, respectively. In scenario 3, the mean D95% was 72.2 (± 0.27) Gy to 18F-miso versus 70.4 (± 0.74) and 69.5Gy (± 0.74) for 18FDG hotspot and per-treatment 18FDG, respectively. In scenario 4, the mean D95% was 73.1 (± 0.3) Gy to 18FDG per-treatment versus 71.9 (± 0.61) and 69.8 (± 0.61) Gy for 18FDG hotspot and 18F-miso, respectively. The dose/volume constraints to OARs were matched in all scenarios.The mean target volumes were 4.8 cc (± 1.1) for 18FDG and 18F-miso PET/CT functional images. The most relevant strategy should be investigated in clinical trials.Escalated doses can be selectively planned in NSCLC target volumes delineated on NCT01576796, registered 15 june 2012)(RTEP5,  The majority of non-metastatic NSCLC cannot undergo surgical resection with curative-intent, either due to the patient’s medical condition or to cancer local extension. Concomitant Radiotherapy - Chemotherapy (RTCT) is the standard curative-intent treatment for non-operable patients/non resectable cancers . Large eer death .18F-miso to identify and delineate hypoxic areas within the 18FDG-defined Gross Target Volume (GTV) [18F-miso uptake. Doses up to 86 Gy could not reverse the poor prognosis features of 18F-miso positive tumours. In this study, hypoxic tumours with boost had the same local control despite twice as large volumes. The patients’ data were used to test in silico three strategies for selective increase in total dose, based on functional imaging. For the strategies with boost, we tested at a dose of 74 Gy to be comparable to the study of Bradley et al. [18FDG metabolic hotspot (70% of SUVmax) (plan 2), 2/ the pre-RT 18F-MISO-affine (SUV > 1.4) volume (plan 3) or the per-treatment 18FDG uptake (40% of SUVmax) (plan 4).In a phase II study , we used y et al.  and our The details of the study can be found elsewhere . Fifty-f18FDG PET/CT (FDG1 and FDG2) and two 18F-miso PET/CT (F-miso1 and F-miso1) acquisitions were performed before and during RT using the same machine and under the identical operational conditions, a centrally supervised quality control securing homogeneity in the image quality in all centres [1 was acquired in treatment position , at least 15 days after the last administration of chemotherapy. F-miso1 was scheduled 48 h after the FDG1. The F-miso PET/CT acquisitions were reviewed by 3 independent experts (out of the 9 experts) who decided upon the presence or absence of uptake [1/F-miso1 and the start of radiotherapy. The F-miso2 was not associated with a planification due to the very low contrast in per-treatment [Two  centres . FDG1 wareatment .1, FDG2 and F-miso1 were co-registered to the planning CT scanner , focusing on the tumour. The GTV for FDG1 was defined as the sum of the voxels with uptake > 40% of the SUVmax inside primary tumour or nodes and corrected from CT data. The 18FDG hotspot was defined as all voxels with uptake > 70% of the SUVmax inside the primary tumour or nodes (BTVm1). The hypoxic volume (BTVh) was defined as the sum of voxels with SUV ≥1.4 on F-miso1 [For each patient, the CTs of FDG F-miso1 , 10. TheFour IMRT by step and shoot scenarios were applied in each patient. All the dose calculations were corrected for heterogeneity  and for the optimization . The total dose was prescribed at ICRU point, the dose delivered in the PTV having to be within 95% and 107% of the prescribed dose. Absolute priority constraints were a maximum dose to the spinal cord < 45 Gy and no more than 30% of the total lung volume (excluding the PTV) receiving ≥20 Gy (V20Gy). The secondary dose/volume constraints were no more than 30% of the oesophagus or the heart receiving ≥50 or ≥ 35 Gy, respectively.The first scenario  was to deliver 66 Gy in the PTV based on FDG1. The experimental scenarios had to deliver 66 Gy in the FDG1 PTV (PTV66) and an additional dose up to 74 Gy in three different smaller target volumes. The boost target volumes were the metabolic hotspot on pre-treatment FDG PET/CT , the hypoxic volume on pre-treatment F-miso PET/CT , and the residual uptake on the FDG PET/CT at 42 Gy . The treatment was planned with a simultaneous boost from the start of the radiotherapy for plan 2 and 3 and from 50 Gy for the plan 4.Descriptive statistics  were calculated for the quantitative variables. Frequency and percentages with 95% confidence intervals (CI) were computed for the qualitative variables. Levene’s test was used to assess variances equality when comparing the quantitative variables means between two or more groups (ANOVA). All statistical calculations were performed with MedCalc Software .The present study was based on 21 patients  = 59 ± 8 years). There were 7 adenocarcinomas, 12 squamous cell carcinomas and 2 undifferentiated carcinomas. The stages distribution was 1 IIB, 12 IIIA and 8 IIIB  smaller than the BTV FDG per-RT  and the BTV hypoxic . Similarly, the mean PTV66Gy was 473 ± 69 cc versus 35 ± 5.8 cc for PTV FDG 70%, 111 ± 24 cc for PTV FDG per-RT, and 105 ± 26 cc for PET F-miso (Table The mean CTV18FDG hotspot), the mean dose to 95% of the PTV (D95%) was 72.5 ((± 0.25) Gy versus 67.9 (± 0.49) (p < 0.0001) in F-miso PTV and 67.9 Gy (± 0.52) (p < 0.0001) in per-treatment 18FDG PTV. In scenario 3 (boost to 18F-miso), the mean D95% was 72.2 (± 0.27) Gy versus 70.4 (± 0.33) (p = 0.74) in 18FDG hotspot and 69.5 (± 0.74) Gy (p = 0.009) in per-treatment 18FDG PTV. In scenario 4 (boost to per-treatment 18FDG), the mean D95% was 73.1 (± 0.3) Gy versus 71.9 (± 0.61) (p = 0.2) in 18FDG hotspot and 69.8 (± 0.61) Gy (p = 0.0001) to FMISO PTV  (Table The dose/volume constraints to the organs at risk were matched without significant differences between scenarios except to the heart between the plan to 66 Gy and FDG per-treatment plan with V35 at respectively 5% (± 1.7) and 5.4% (± 1.8)  and improved radiotherapy delivery (IMRT) opens the way to selectively increase total dose in biologically-relevant parts of the tumour. In a previous study, we showed that areas of high 18FDG uptake (SUV > 70% SUVmax) on pre-treatment PET/CT scans were associated to tumour areas at greater risk of relapse [max volume delineated on pre-treatment 18FDG PET/CT . We are presently conducting a phase II study  where the radiotherapy dose is escalated up to 74 Gy in the metabolic residual as assessed on FDG-PET/CT performed at 42 Gy. Targeting the hypoxic volume as identified by 18F-miso PET/CT before or during radiotherapy was investigated in RTEP5 [RTCT is the reference treatment for locally advanced non operable NSCLC. The high incidence of relapse within the target volume suggest insufficient total doses of radiotherapy to achieve local control. The RTOG 0617 randomized trial  demonstr relapse . Similar relapse  and in o relapse . A Europin RTEP5 , 15.18FDG uptake is associated to the presence of hypoxia via the upregulation of glucose transporter 1 by hypoxia-inducible factor 1 [18FDG and 18F-miso provide different and possibly complementary information. Given the impact on dose distribution as observed here, the selection of the most relevant strategy will rely on clinical trials. In our RTEP5 phase II [The three scenarios investigated here yielded different target volumes for radiotherapy dose escalation. Our data contradict the idea that factor 1 , 17. 18Fphase II .Non respiratory-gated images for planning CT and PET/CT could have hampered the delineation precision and strategy for reducing the PTV margins. When the RTEP5 study was initiated, 4D acquisitions (and IMRT) were not routinely performed in the participating centres. The decision was made to require ungated acquisitions  in order to secure our recruitment objectives and the exportability of our results. The majority of our patients had large stage III tumours involving the mediastinum, limiting breathing movements. In addition, our delineation criteria on 18F-miso PET/CT was validated in free-breathing patients [18F-miso uptake was associated to worse prognosis in our previous RTEP5 study [18F-miso uptake, were not eligible to the present planning study, mostly because of too large target volumes precluding dose escalation without compromising OAR tolerance. Beyond feasibility studies, randomised trials are warranted to demonstrate the value of radiotherapy assisted by functional imaging.Patient selection is another issue. Positive P5 study . Ten pat18FDG and pre-treatment 18F-miso PET/CT yield different candidate target volumes for selective increase in radiotherapy dose in patients with NSCLC. Our in-silico study shows that IMRT provides radiotherapy plans matching the pre-defined dose/volume objectives and constraints. Clinical trials are required to select the relevant strategies to improve outcome after concomitant chemo-radiotherapy.Pre−/per-treatment