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News Article | April 7, 2017
Site: www.techtimes.com

The evils of smoking are well-known and even prompted California to impose a price hike on cigarettes to deter people from the unhealthy habit. Now, a new study reveals that smoking led to over 11 percent deaths all over the world. According to the study, nearly 1 billion individuals all over the world smoke a cigarette each day. An alarming statistic that came to the fore was that in 2015, 1 out of 10 deaths occurred because of smoking. In 2015, 11.5 percent global deaths were attributed to smoking. Surprisingly, 52.2 percent of the deaths caused due to smoking, occurred in four countries, including the United States. Apart from the United States, the other countries to make it to the top four for deaths caused due to smoking were — China, India, and Russia. Smoking has claimed more than 5 billion lives since 1990. The negative impact of smoking is growing at an alarming rate, especially in countries falling under low income groups. To ascertain the prevalence of smoking and its global effects, the researchers analyzed data on smokers from 195 territories and countries. This data was from 1990 to 2015. "We synthesised 2818 data sources with spatiotemporal Gaussian process regression and produced estimates of daily smoking prevalence by sex, age group, and year for 195 countries and territories from 1990 to 2015," noted the researchers. The researchers found that there was decline in the percentage of smokers globally. However, owing to population growth, the number of smokers on a whole, had increased all around the world. This implies that as the number of people inhabiting Earth rises, the number of smokers grow as well. However, if the number of smokers are compared to the total population, the percentage is lesser than what it was over two decades years ago. "Growth in the sheer number of daily smokers still outpaces the global decline in daily smoking rates, indicating the need to prevent more people from starting the tobacco habit and to encourage smokers to quit," said Emmanuela Gakidou, senior author of the study. The researchers discovered that in 2015, roughly 933 million people smoked every single day and 80 percent of these smokers were men. However, from 1990 to 2015, there was a worldwide decline in smoking prevalence from 29.4 percent to 15.3 percent. The researchers found that in 2015, one in every four men worldwide smoked every day. The statistic for women is a shade better as it was found that 1 in every 20 women smoked on a daily basis. In this period, the daily smoking rate for men fell from 35 percent in 1990 to 25 percent in 2015. Comparatively, the daily smoking rate for women reduced from 8 percent in 1990 to 5 percent in 2015. Among the top four countries having the highest death rate due to smoking, China accounts for nearly 254 million male smokers and India follows close behind, accounting for 91 million male smokers. The United States led the countries with the most female smokers, accounting for 17 million female smokers in 2015. It was followed by China and India with 14 million and 13.5 million female smokers, respectively. However, in 2015, the maximum rate of female smoking was in Greenland as 44 percent of women in the region smoked daily. Gakidou stresses on the importance of tobacco control programs. He urges everyone around the world to take the implications of smoking on one's health seriously. The study has been published in journal The Lancet on April 5. © 2017 Tech Times, All rights reserved. Do not reproduce without permission.


News Article | April 28, 2017
Site: www.cemag.us

A “chemical imaging” system that uses a special type of laser beam to penetrate deep into tissue might lead to technologies that eliminate the need to draw blood for analyses including drug testing and early detection of diseases such as cancer and diabetes. The system, called stimulated Raman projection microscopy and tomography, makes possible “volumetric imaging” without using fluorescent dyes that might affect biological functions and hinder accuracy, said Ji-Xin Cheng, a professor in Purdue University’s Weldon School of Biomedical Engineering, Department of Chemistry and Birck Nanotechnology Center. “Volumetric chemical imaging allows a better understanding of the chemical composition of three-dimensional complex biological systems such as cells,” he said. The technology uses a type of laser beam called a Bessel beam, which maintains focus for a longer distance than a traditional “Gaussian beam” used in other imaging technologies, making it possible to penetrate deep into tissue. Stimulated Raman spectroscopy eliminates the need for fluorescent dyes. The technology yields more accurate data than other methods because it allows imaging of the entire cell by “adding up” signals produced from the scanning beam, Cheng said. Because the Bessel beam makes possible deep-tissue imaging, it could lead to systems that eliminate the need to draw blood for analyses such as drug testing and detection of biomarkers for non-invasive early diagnosis of diseases, Cheng said. “This is a long-term goal,” he said. “In the meantime, much more research is needed to improve the system.” The researchers proved the concept by imaging fat storage in living cells. Findings are detailed in a research paper appearing on April 24 in the journal Nature Communications. The reported technology yields information about chemical composition, collecting a series of images while rotating the sample and reconstructing the 3-D structure through image reconstruction algorithms. The Bessel beam is produced using a pair of cone-shaped “axicon” lenses and is combined with a microscope objective. Its use for volumetric fluorescence imaging was previously demonstrated by physicist Eric Betzig, who won the Nobel Prize in chemistry in 2014 for his pioneering contribution to super-resolution fluorescence microscopy. Super-resolution technology allows researchers to resolve structural features far smaller than the wavelength of visible light, sidestepping the “diffraction limit” that normally prevents imaging of features smaller than about 250 nanometers, which is large compared to certain biological molecules and structures in cells. However, fluorescence microscopy usually requires the use of fluorescent tags, which may interfere with biological processes and hinder accuracy for determining chemical structure. Future research will include work to increase the detection sensitivity of the system and improve the imaging quality and speed. “There is plenty of room for improvement,” Cheng said. “The system is based on a bulky and relatively expensive femtosecond laser, which limits its potential for broad use and clinical translation. Nevertheless, we anticipate that this limitation can be circumvented through engineering innovations to reduce the cost and size of our technology. We also note that the Bessel beam can be produced using fibers, which could simplify the system and enable endoscopic applications.” The paper was authored by Xueli Chen, a visiting scholar from Xidian University in China; Purdue postdoctoral research associate Chi Zhang; Purdue doctoral students Peng Lin and Kai-Chih Huang; Xidian University researchers Jimin Liang and Jie Tian; and Cheng. The research was supported by funds from the Keck Foundation and National Institutes of Health.


News Article | April 27, 2017
Site: phys.org

The system, called stimulated Raman projection microscopy and tomography, makes possible "volumetric imaging" without using fluorescent dyes that might affect biological functions and hinder accuracy, said Ji-Xin Cheng, a professor in Purdue University's Weldon School of Biomedical Engineering, Department of Chemistry and Birck Nanotechnology Center. "Volumetric chemical imaging allows a better understanding of the chemical composition of three-dimensional complex biological systems such as cells," he said. The technology uses a type of laser beam called a Bessel beam, which maintains focus for a longer distance than a traditional "Gaussian beam" used in other imaging technologies, making it possible to penetrate deep into tissue. Stimulated Raman spectroscopy eliminates the need for fluorescent dyes. The technology yields more accurate data than other methods because it allows imaging of the entire cell by "adding up" signals produced from the scanning beam, Cheng said. Because the Bessel beam makes possible deep-tissue imaging, it could lead to systems that eliminate the need to draw blood for analyses such as drug testing and detection of biomarkers for non-invasive early diagnosis of diseases, Cheng said. "This is a long-term goal," he said. "In the meantime, much more research is needed to improve the system." The researchers proved the concept by imaging fat storage in living cells. Findings are detailed in a research paper appearing on April 24 in the journal Nature Communications. The reported technology yields information about chemical composition, collecting a series of images while rotating the sample and reconstructing the 3-D structure through image reconstruction algorithms. The Bessel beam is produced using a pair of cone-shaped "axicon" lenses and is combined with a microscope objective. Its use for volumetric fluorescence imaging was previously demonstrated by physicist Eric Betzig, who won the Nobel Prize in chemistry in 2014 for his pioneering contribution to super-resolution fluorescence microscopy. Super-resolution technology allows researchers to resolve structural features far smaller than the wavelength of visible light, sidestepping the "diffraction limit" that normally prevents imaging of features smaller than about 250 nanometers, which is large compared to certain biological molecules and structures in cells. However, fluorescence microscopy usually requires the use of fluorescent tags, which may interfere with biological processes and hinder accuracy for determining chemical structure. Future research will include work to increase the detection sensitivity of the system and improve the imaging quality and speed. "There is plenty of room for improvement," Cheng said. "The system is based on a bulky and relatively expensive femtosecond laser, which limits its potential for broad use and clinical translation. Nevertheless, we anticipate that this limitation can be circumvented through engineering innovations to reduce the cost and size of our technology. We also note that the Bessel beam can be produced using fibers, which could simplify the system and enable endoscopic applications." The paper was authored by Xueli Chen, a visiting scholar from Xidian University in China; Purdue postdoctoral research associate Chi Zhang; Purdue doctoral students Peng Lin and Kai-Chih Huang; Xidian University researchers Jimin Liang and Jie Tian; and Cheng. Explore further: Imaging uses 'photothermal effect' to peer into living cells More information: Xueli Chen et al. Volumetric chemical imaging by stimulated Raman projection microscopy and tomography, Nature Communications (2017). DOI: 10.1038/ncomms15117


News Article | May 10, 2017
Site: www.nature.com

HEK293 cells stably transfected with the STF plasmid encoding the firefly luciferase reporter under the control of a minimal promoter, and a concatemer of 7 LEF/TCF binding sites32, were obtained from J. Nathans. Mouse L cells stably transfected with the STF plasmid and a constitutively expressed Renilla luciferase (control reporter) were obtained from C. Kuo. L cells transfected with a mouse WNT3A expression vector to produce conditioned media were obtained from the ATCC. A375, SH-SY5Y and A549 cells were stably transfected with the BAR plasmid encoding the firefly luciferase reporter under the control of a minimal prompter and a concatemer of 12 TCF/LEF binding sites and a constitutively expressed Renilla luciferase (control reporter) using a lentiviral-based approach33. All reporter cell lines were cultured in complete DMEM medium (Gibco) supplemented with 10% FBS, 1% penicillin, streptomycin, and l-glutamine (Gibco), at 37 °C and 5% CO and cultured in the presence of antibiotics for selection of the transfected reporter plasmid. C3H10T1/2 cells were obtained from the ATCC. Human primary MSCs were obtained from Cell Applications, Inc. Mouse primary MSCs were obtained from Invitrogen. Cell lines have not been tested for mycoplasma contamination. The coding sequence of B12 containing a C-terminal 6×His-tag was cloned into the pET28 vector (Novagen) for bacterial cytoplasmic protein expression. Protein expression was performed in transformed BL21 cells, expression was induced with 0.7 mM IPTG at an OD   of 0.8 for 3–4 h. Cells were pelleted, lysed by sonication in lysis buffer (20 mM HEPES, pH 7.2, 300 mM NaCl, 20 mM imidazole), and soluble fraction was applied to Ni-NTA agarose (QIAGEN). After washing the resin with lysis buffer containing 500 mM NaCl, B12 was eluted with 300 mM imidazole, and subsequently purified on a Superdex 75 size-exclusion column (GE Healthcare) equilibrated in HBS (10 mM HEPES, pH 7.2, 150 nM NaCl). XWnt8 was purified from a stably transfected Drosophila S2 cell line co-expressing XWnt8 and mouse FZD8 CRD–Fc described previously4. Cells were cultured in complete Schneider’s medium (Thermo Fisher Scientific), containing 10% FBS and supplemented with 1% l-glutamine, penicillin and streptomycin (Gibco), and expanded in Insect-Xpress medium (Lonza). A complex of XWnt8 and FZD8 CRD–Fc was captured from the conditioned media on Protein A agarose beads (Sigma). After washing with 10 column volumes of HBS, XWnt8 was eluted with HBS containing 0.1% n-dodecyl-β-d-maltoside (DDM) and 500 mM NaCl, while the FZD8 CRD–Fc remained bound to the beads. All other proteins were expressed in High Five (Trichoplusia ni) cells (Invitrogen) using the baculovirus expression system. To produce the B12-based surrogate, the coding sequences of B12, a flexible linker peptide comprising of 0, 1, 2 or 3 GSGSG-linker repeats, followed by the C-terminal domain of human DKK1 (residues 177–266), and a C-terminal 6×His-tag, were cloned into the pAcGP67A vector (BD Biosciences). To clone the scFv-based surrogate ligand, the sequence of the Vantictumab was retrieved from the published patent, reformatted into a scFv, and cloned at the N terminus of the surrogate variant containing the GSGSG linker peptide. To produce recombinant FZD CRD for crystallization, surface plasmon resonance measurements, SEC-MALS experiments and functional assays, the CRDs of human FZD1 (residues 113–182), human FZD4 (residues 42–161), human FZD5 (residues 30–150), human FZD7 (residues 36–163), human FZD8 (residues 32–151) and human FZD10 (residues 30–150), containing a C-terminal 3C protease cleavage site (LEVLFQ/GP), a biotin acceptor peptide (BAP)-tag (GLNDIFEAQKIEWHE) and a 6×His-tag were cloned into the same vector. The human FZD8 CRD used for crystallization contained only a C-terminal 6×His-tag, in addition to a Asn49Gln mutation to mutate the N-linked glycosylation site. FZD1/FZD8 CRD for inhibition assay contained a C-terminal 3C protease cleavage site, Fc-tag (constant region of human IgG), and a 6×His-tag. Human DKK1 (residues 32–266) with a C-terminal BAP-tag and 6×His-tag, and the two furin-like repeats of human RSPO2 (residues 36–143) with a N-terminal Fc-tag and a C-terminal 6×His-tag, were cloned also into the pAcGP67A vector. All proteins were secreted from High Five insect cells grown in Insect-Xpress medium, and purified using Ni-NTA affinity purification, and size-exclusion chromatography equilibrated in HBS (10 mM HEPES, pH 7.3, 150 nM NaCl). Enzymatic biotinylation was performed in 50 mM bicine, pH 8.3, 10 mM ATP, 10 mM magnesium acetate, 0.5 mM d-biotin with recombinant glutathione S-transferase (GST)-tagged BirA ligase overnight at 4 °C, and proteins were subsequently re-purified on a Superdex 75 size-exclusion column to remove excess biotin. We attempted to mimic the native Wnt–FZD lipid–protein interaction with a de novo designed protein–protein binding interface. A 13-residue alanine helix was docked against the lipid-binding cleft using Foldit34. This structural element was grafted onto a diverse set of native helical proteins using the Rosetta Epigraft35 application to discover scaffolds with compatible, shape-complementary backbones. Prototype designs were selected by interface size and optimized using RosettaScripts36 to perform side-chain redesign. 50 selected designs were further manually designed to ensure charge complementarity and non-essential mutations were reverted to the wild-type amino acid identity to maximize stability. DNA was obtained from Gen9 and screened for binding via yeast surface display as previously described with 1 μM biotinylated FZD8 CRD pre-incubated with 025 µM SAPE (Life Technologies)37. A design based on the scaffold with PDB code 2QUP, a uncharacterized four-helix bundle protein from Bacillus halodurans, demonstrated binding activity under these conditions, whereas knockout mutants Ala52Arg and Ala53Asd made using the Kunkel method38 abrogated binding, verifying that the functional interface used the predicted residues. Wild-type scaffold 2QUP did not bind, confirming that activity was specifically due to design. To improve the affinity of the original design, a full-coverage site-saturation mutagenesis library was constructed for design based on the 2QUP scaffold via the Kunkel mutagenesis method38 using forward and reverse primers containing a ‘NNK’ degenerate codon and 21-bp flanking regions (IDT). A yeast library was transformed as previously described39 and sorted for three rounds, collecting the top 1% of binders using the BD Influx cell sorter. Naive and selected libraries were prepared and sequenced, and the data was processed as previously described37 using a Miseq (Illumina) according to manufacturer protocols. The most enriched 11 mutations were identified by comparison of the selected and unselected pools of binders and were combined in a degenerate library containing all enriched and wild-type amino acid identities at each of these positions. This combination library was assembled from the oligonucleotides (IDT) listed below for a final theoretical diversity of around 800 k distinct variants. This library was amplified, transformed, and selected to convergence over five rounds, yielding the optimized variant B12. The B12–FZD8 CRD(N49Q) complex was formed by mixing purified B12 and FZD8 CRD(N49Q) in stoichiometric quantities. The complex was then treated with 1:100 (w/w) carboxypeptidase A (Sigma) overnight at 4 °C, and purified on a Superdex 75 (GE Healthcare Life Sciences) size-exclusion column equilibrated in HBS. Purified complex was concentrated to around 15 mg ml−1 for crystallization trials. Crystals were grown by hanging-drop vapour diffusion at 295 K, by mixing equal volumes of the complex and reservoir solution containing 42–49% PEG 400, 0.1 M Tris, pH 7.8–8.2, 0.2 M NaCl, or 20% PEG 3000, 0.1 M sodium citrate, pH 5.5. While the PEG 400 condition is already a cryo-protectant, the crystals grown in the PEG 3000 condition were cryoprotected in reservoir solution supplemented with 20% glycerol before flash freezing in liquid nitrogen. Crystals grew in space groups P2 (PEG 400 condition) and P2 (PEG 3000 condition), respectively, with 2 and 4 complexes in the asymmetric units. Cell dimensions are listed in Supplementary Table 1. Data were collected at beamline 8.2.2 at the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory. All data were indexed, integrated, and scaled with the XDS package40. The crystal structures in both space groups were solved by molecular replacement with the program PHASER41 using the structure of the FZD8 CRD (PDB code 1IJY) and the designed model of a minimal core of B12 as search models. Missing residues were manually build in COOT42 after initial rounds of refinement. Several residues at the N terminus (residues 1 to 16/17/20/21), at the C terminus (residues after 117) and several residues within loop regions were unstructured and could not been modelled. Furthermore, we observed that in both crystal forms, B12 underwent domain swapping, and one B12 molecule lent helix 3 and 2 to another B12, resulting in a closely packed B12 homodimer. The density of the loops connecting helixes 1 and 2, and 3 and 4 were clearly visible, and folded into helical turns. Yet, SEC-MALS experiments confirmed that B12 existed as a monomer in solution, and complexed FZD8 CRD with a 1:1 stoichiometry. PHENIX Refine43 was used to perform group coordinate refinement (rigid body refinement), followed by individual coordinate refinement using gradient-driven minimization applying stereo-chemical restraints, NCS restraints, and optimization of X-ray/stereochemistry weight, and individual B-factor refinement. Initial rounds of refinement were aided by restraints from the high-resolution mouse FZD8 CRD structure as a reference model. Real space refinement was performed in COOT into a likelihood-weighted SigmaA-weighted 2mF  − DF map calculated in PHENIX. The final model in the P2 space group was refined to 3.20 Å with R and R values of 0.2002 and 0.2476, respectively (Supplementary Table 1). The quality of the structure was validated with MolProbity44. 99.5% of residues are in the favoured region of the Ramachandran plot, and no residue in the disallowed region. The structure within the P2 space group was refined to 2.99 Å with R and R values of 0.2253 and 0.2499, respectively, with 99.2% of residues in the favoured region of the Ramachandran plot, and no residues in the disallowed region. See Supplementary Table 1 for data and refinement statistics. Structure figures were prepared with the program PYMOL. Binding measurements were performed by surface plasmon resonance on a BIAcore T100 (GE Healthcare) and all proteins were purified on SEC before experiments. Biotinylated FZD1 CRD, FZD5 CRD, FZD7 CRD and FZD8 CRD were coupled at a low density to streptavidin on a SA sensor chip (GE Healthcare). An unrelated biotinylated protein was captured at equivalent coupling density to the control flow cells. Increasing concentrations of B12 and scFv–DKK1c were flown over the chip in HBS-P (GE Healthcare) containing 10% glycerol and 0.05% BSA at 40 μl ml−1. The chip surface was regenerated after each injection with 2 M MgCl in HBS-P or 50% ethylene glycol in HBS-P (scFv–DKK1c measurements), or 4 M MgCl in HBS-P (B12 measurements) for 60 s. Curves were reference-subtracted and all data were analysed using the Biacore T100 evaluation software version 2.0 with a 1:1 Langmuir binding model to determine the K values. To characterize the FZD-specificity of B12, the yeast display vector encoding B12 was transformed into EBY100 yeast. To induce the display of B12 on the yeast surface, cells were growing in SGCAA medium45, 46 for 2 days at 20 °C. 1 × 106 yeast cells per condition were washed with PBE (PBS, 0.5% BSA, 2 mM EDTA), and stained separately with 0.06–1,000 nM biotinylated FZD1/4/5/7/8/10 CRDs for 2 h at 4 °C. After washing twice with ice-cold PBE, bound FZD CRDs were labelled with 10 nM strepdavidin-Alexa647 for 20 min. Cells were fixed with 4% paraformaldehyde, and bound FZD CRD was analysing on an Accuri C6 flow cytometer. FZD8 fused to an N-terminal HaloTag47 and LRP6 fused to an N-terminal SNAP-tag48 were cloned into the pSEMS-26m vector (Covalys Biosciences) by cassette cloning49, 50. The template pSEMS-26m vectors had been coded with DNA sequences of the SNAP-tag or the HaloTag, respectively, together with an Igκ leader sequence (from the pDisplay vector, Invitrogen) as described previously50. The genes of full-length mouse Fzd8 or human LRP6 without the N-terminal signal sequences were inserted into pSEMS-26m via the XhoI and AscI or AscI and NotI, restriction sites, respectively. A plasmid encoding a model transmembrane protein, maltose-binding protein fused to a transmembrane domain, fused to an N-terminal HaloTag was prepared as described recently13. HeLa cells were cultivated at 37 °C, 5% CO in MEM Earle’s (Biochrom AG, FG0325) supplemented with 10% fetal calf serum and 1% nonessential amino acids. Cells were plated in 60-mm cell culture dishes to a density of 50% confluence and transfected via calcium phosphate precipitation49. 8–10 h after transfection, cells were washed twice with PBS and the medium was exchanged, supplied with 2 μM porcupine inhibitor IWP-2 for inhibiting maturation of endogenous Wnt in HeLa cells51. 24 h after transfection, cells were plated on glass coverslips pre-coated with PLL-PEG-RGD52 for reducing nonspecific binding of dyes during fluorescence labelling. After culturing for 12 h, coverslips were mounted into microscopy chambers for live-cell imaging. SNAP-tag and HaloTag were labelled by incubating cells with 50 nM benzylguanine-DY649 (SNAP-Surface 649, New England Biolabs) and 80 nM of HaloTag tetramethylrhodamine ligand (HTL-TMR, Promega) for 20 min at 37 °C. Under these conditions, effective degrees of labelling estimated from single molecule assays with a HaloTag–SNAP-tag fusion protein were ~40% for the SNAP-tag and ~25% for the HaloTag13. After washing three times with PBS, the chamber was refilled with MEM containing 2 μM IWP-2 for single-molecule fluorescence imaging. Single-molecule fluorescence imaging was carried out by using an inverted microscope (Olympus IX71) equipped with a triple-line total internal reflection (TIR) illumination condenser (Olympus) and a back-illuminated EMCCD camera (iXon DU897D, 512 × 512 pixel from Andor Technology). A 561-nm diode solid state laser (CL-561-200, CrystaLaser) and a 642-nm laser diode (Luxx 642-140, Omicron) were coupled into the microscope for excitation. Laser lights were reflected by a quad-line dichroic beam splitter (Di R405/488/561/647, Semrock) and passed through a TIRF objective (UAPO 150×/1.45, Olympus). For simultaneous dual-colour detection, a DualView microimager (Optical Insight) equipped with a 640 DCXR dichroic beamsplitter (Chroma) in combination with bandpass filters FF01-585/40 and FF01 670/30 (Semrock), respectively, was mounted in front of the camera. The overlay of the two channels was calibrated by imaging fluorescent microbeads (TetraSpeck microspheres 0.1 μm, T7279, Invitrogen), which were used for calculating a transformation matrix. After channel alignment, the deviation between the channels was below 10 nm. For single-molecule imaging, typical excitation powers of 1 mW at 561 nm and 0.7 mW at 642 nm measured at the objective were used. Time series of 150–300 frames were recorded at 30 Hz (4.8–9.6 s). An oxygen scavenging system containing 0.5 mg ml−1 glucose oxidase, 40 mg ml−1 catalase, and 5% (w/v) glucose, together with 1 μM ascorbic acid and 1 μM methyl viologene, was added to minimize photobleaching53. Receptor dimerization was initiated by incubating with 100 nM Wnt proteins or surrogates. Images were acquired after 5 min incubation in the presence of the ligands. All live-cell imaging experiments were carried out at room temperature. A 2D Gaussian mask was used for localizing single emitters54, 55. For colocalization analysis to determine the heterodimerization fraction, particle coordinates from two channels were aligned by a projective transformation (cp2tform of type ‘projective’, MATLAB 2012a) according to the transformation matrix obtained from microbead calibration measurement. Particles colocalized within a distance of 150 nm were selected. Only co-localized particles, which could be tracked for at least 10 consecutive frames (that is, molecules co-locomoting for at least 0.32 s) were accepted as receptor heterodimers or hetero-oligomers, which has been previously found to be a robust criterion for protein dimerization13. The fraction of heterodimerization or hetero-oligomerization was determined as the number of co-locomotion trajectories with respect to the number of the receptor trajectories. Since the receptor expression level of FZD8 or LRP6 was variable in the transiently transfected cells, only cells with similar receptor expression levels were considered (less than three times the excess of one subunit over the other). The smaller number of trajectories of either FZD8 or LRP6 was regarded as the limiting factor and therefore taken as a reference for calculating the heterodimerized/hetero-oligomerized fraction. Oligomerization values were not corrected for the degree of labelling. Single-molecule trajectories were reconstructed using the multi-target tracing (MTT) algorithm56. The detected trajectories were evaluated with respect to their step length distribution to determine the diffusion coefficients. For a reliable quantification of local mobilities, we estimated diffusion constants from the displacements with three frames (96 ms). Step-length histograms were obtained from all single molecule trajectories and fitted by a two-component model of Brownian diffusion, thus taking into account the intrinsic heterogeneity of protein diffusion in the plasma membrane57, 58. A bimodal probability density function p(r) was used for a nonlinear least square fit of the step-length histogram: where is the percentage of the fraction, contains the diffusion coefficient of each fraction (nδt = 96 ms). Average diffusion coefficients were determined by weighting the diffusion coefficients with the corresponding fractions. Single-molecule intensity distribution of individual diffraction-limited spots was extracted from the first 50 images of the recorded time lapse image sequence, in which photobleaching of dyes was kept below 10%13. Oligomerization of receptors was evaluated by fitting the obtained single molecule intensity with a multi-component Gaussian distribution function59. To ensure a reliable analysis, monomeric receptors were first distinguished based on the observation that monomers diffused much faster than oligomers. Therefore, the characteristic intensity distribution of monomeric receptor subunits was obtained by tracking of the fast mobile fraction. Fractions of the monomer, dimer, trimer and higher oligomers were then de-convoluted from the single molecule intensity distribution, presuming that intensities of clusters were multiples of the monomer intensity distribution. Immortal cells were seeded in triplicate for each condition in 96-well plates, and stimulated with surrogates, XWnt8, WNT3A conditioned media, control proteins, or other treatments for 20–24 h. After washing cells with PBS, cells in each well were lysed in 30 μl passive lysis buffer (Promega). 10 μl per well of lysate was assayed using the Dual Luciferase Assay kit (Promega) and normalized to the Renilla luciferase signal driven constitutively by the human elongation factor-1 alpha promoter to account for cell variability. A375 BAR, SH-SY5Y BAR, L STF and HEK293 STF cells were plated at a density of 10,000–20,000 cells per well, and treatment was started after 24 h in fresh medium. A549 BAR cells were plated at a density of 5,000 cells per well in the presence of 2 μM IWP-2 (Calbiochem) to suppress endogenous Wnt secretion, and treatment was started after 48 h in fresh medium containing fresh IWP-2. To induce β-catenin accumulation, SH-SY5Y BAR cells were treated for 2 h with scFv–DKK1c, WNT3A conditioned media (positive control), B12 (negative control protein) and mock conditioned media (from untransfected L cells, negative control) at 37 °C, 5% CO . After, cells were washed twice with PBS. For β-catenin stabilization assay, cells were scraped into hypotonic lysis buffer (10 mM Tris-HCl pH 7.4, 0.2 mM MgCl , supplemented with protease inhibitors), incubated on ice for 10 min, and homogenized using a hypodermic needle. Sucrose and EDTA were added to final concentration of 0.25 M and 1 mM, respectively. For LRP6 phosphorylation assay, cells were lysed in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.5% sodium deoxylate, 1% Triton X-100), supplemented with protease inhibitor and phosphatase inhibitor for 1 h at 4 °C. Lysates were centrifuged at 12,000g for 1 h at 4 °C. Supernatants were then diluted into SDS sample buffer. For immunoblotting, samples were resolved on a 12% Mini-PROTEAN(R)TGX precast protein gel (Bio-Rad) and transferred to a PVDF membrane. The membranes were cut horizontally approx. at the 64 kDa mark of the SeeBlue plus 2 molecular mass marker (Invitrogen). Top half of the blot was incubated with anti-β-catenin primary antibody ((D10A8)XP, rabbit, Cell Signaling 8480), LRP6 antibody ((C47E12), rabbit, Cell Signaling 3395), and P-LRP6 (S1490) antibody (rabbit, Cell Signaling 2568), and the bottom part with the anti-α-tubulin primary antibody (mouse, DM1A, Sigma) in PBS containing 0.1% Tween-20 and 5% BSA overnight at 4 °C. Blots were then washed, incubated with the corresponding secondary antibodies in the same buffer, before washing and developing using the ECL prime western blotting detection reagent (GE Healthcare). To induce β-catenin accumulation, K562 and cells were stimulated for 0, 15, 30, 45, 60, 90 and 120 min with 10 nM scFv–DKK1c, recombinant Wnt3a (R&D Systems), B12 (negative control protein) or plain complete growth medium at 37 °C, 5% CO . After, cells were washed twice with PBS, fixed with 4% PFA for 10 min at room temperature, and permeabilized in 100% methanol for at least 30 min at −80 °C. The cells were than stained with Alexa-647 conjugated anti-β-catenin antibody (L54E2) (Cell Signaling Technology, 1:100–1: 50 dilution). Fluorescence was analysed on an Accuri C6 flow cytometer. Total RNA was isolated using either TRIZOL (Invitrogen) or RNeasy plus micro kit (QIAGEN) according to manufacturer’s protocols. A total of 2 μg RNA were used to generate cDNA using the RevertAid RT kit (Life Technologies) using oligo(dT)18 mRNA primers (Life Technologies) according to manufacturer’s protocol. 12 ng of cDNA per reaction were used. qPCR was performed using SYBR Green-based detection (Applied Biosystems) according to the manufacturer’s protocol on a StepOnePlus real-time PCR system (ThermoFisher Scientific). All primers were published, or validated by us. Transcript copy numbers were normalized to GAPDH for each sample, and fold induction compared to control was calculated. The following gene-specific validated primers were used: human FZD1: F: 5′-ATCCTGTGTGCTCCTCTTTTGG-3′, R: 5′-GATTGCTTTTCTCCTCTTCTTCAC-3′; human FZD2: F: 5′-CTGGGCGAGCGTGATTGT-3′, R: 5′-GTGGTGACAGTGAAGAAGGTGGAAG-3′; human FZD3: F: 5′-TCTGTATTTTGGGTTGGAAGCA-3′, R: 5′-CGGCTCTCATTCACTATCTCTTT-3′; human FZD4: F: 5′-TGGGCACTTTTTCGGTATTC-3′, R: 5′-TGCCCACCAACAAAGACATA-3′; human FZD5: F: 5′-CCATGATTCTTTAAGGTGAGCTG-3′, R: 5′-ACTTATTCAAGACACAACGATGG-3′; human FZD6: F: 5′-CGATAGCACAGCCTGCAATA-3′, R: 5′-ACGGTGCAAGCCTTATTTTG-3′; human FZD7: F: 5-TACCATAGTGAACGAAGAGGA-3′, R: 5′-TGTCAAAGGTGGGATAAAGG-3′; human FZD8: F: 5′-ACCCAGCCCCTTTTCCTCCATT-3′, R: 5′-GTCCACCCTCCTCAGCCAAC-3′; human FZD9: F: 5′-GCTGTGACTGGAATAAACCCC, R: 5′-GCTCTGCTTACAAGAAAGACTCC-3′; human FZD10: F: 5′-CTCTTCTCTGTGCTGTACACC, R: 5′-GTCTTGGAGGTCCAAATCCA-3′; mouse Fzd1: F: 5′-GCGACGTACTGAGCGGAGTG, R: 5′-TGATGGTGCGGATGCGGAAG-3′60; mouse Fzd2: F: 5′-CTCAAGGTGCCGTCCTATCTCAG, R: GCAGCACAACACCGACCATG-3′60; mouse Fzd3: F: 5′-GGTGTCCCGTGGCCTGAAG-3′, R: 5′-ACGTGCAGAAAGGAATAGCCAAG-3′60; mouse Fzd4: F: 5′-GACAACTTTCACGCCGCTCATC-3′, R: 5′-CAGGCAAACCCAAATTCTCTCAG-3′60; mouse Fzd5: F: 5′-AAGCTGCCTTCGGATGACTA-3′, R: 5′-TGCACAAGTTGCTGAACTCC-3′60; mouse Fzd6: F: 5′-TGTTGGTATCTCTGCGGTCTTCTG-3′, R: 5′-CTCGGCGGCTCTCACTGATG-3′60; mouse Fzd7: F: 5′-ATATCGCCTACAACCAGACCATCC-3′, R: 5′-AAGGAACGGCACGGAGGAATG-3′60; mouse Fzd8: F: 5′-GTTCAGTCATCAAGCAGCAAGGAG-3′, R: 5′-AAGGCAGGCGACAACGACG-3′60; mouse Fzd9: F: 5′-ATGAAGACGGGAGGCACCAATAC-3′, R: 5′-TAGCAGACAATGACGCAGGTGG-3′60; mouse Fzd10: F: 5′-ATCGGCACTTCCTTCATCCTGTC-3′, R: 5′-TCTTCCAGTAGTCCATGTTGAG-3′60; human AXIN2: F: 5′-CTCCCCACCTTGAATGAAGA-3′, R: 5′-TGGCTGGTGCAAAGACATAG-3′; human GAPDH: F: 5′-TGAAGGTCGGAGTCAACGGA-3′, R: 5′-CCATTGATGACAAGCTTCCCG-3′; mouse Gapdh: F: 5′-CCCCAATGTGTCCGTCGTG-3′, R: 5′-GCCTGCTTCACCACCTTCT-3′. Differentiation of C3H10T1/2, and human and mouse primary MSCs were performed essentially as described previously61. In brief, approximately 10,000 cells cm−2 were plated in normal culture medium (αMEM + FBS + penicillin/streptomycin), and allowed to adhere overnight. The following day, the medium was replaced with osteogenic medium (αMEM, 10% FBS, 1% penicillin/streptomycin, 50 μg ml−1 ascorbic acid, 10 mM β-glycerol phosphate (βGP), and replaced every other day. To determine alkaline phosphatase enzymatic activity, cells were fixed for 10 min with 10% formalin in PH7 PBS, before incubation in NBT-BCIP solution (1-Step(tm) NBT/BCIP Substrate Solution (Thermo Fisher Scientific, 34042) for 30 min. qPCR reactions were done with the SYBR method using the following primers: human ACTB F: 5′-GTTGTCGACGACGAGCG-3′, R: 5′-GCACAGAGCCTCGCCTT-3′; human ALPL: F: 5′-GATGTGGAGTATGAGAGTGACG-3′, R: 5′-GGTCAAGGGTCAGGAGTTC-3′; mouse Alpl: F: 5′-AAGGCTTCTTCTTGCTGGTG-3′, R: 5′-GCCTTACCCTCATGATGTCC-3′; mouse Actb: F: 5′-GGAATGGGTCAGAAGGACTC-3′, R: 5′-CATGTCGTCCCAGTTGGTAA-3′; mouse Col2a1 F: 5′-GTGGACGCTCAGGAGAAACA-3′, R: 5′-TGACATGTCGATGCCAGGAC-3′. P26N, normal adult human colon organoids, were established from a tumour-free colon segment of a patient diagnosed with CRC as described18, 62, 63. CFTR-derived colorectal organoids were obtained from a patient at Wilhelmina Children’s Hospital WKZ-UMCU. Informed consent for the generation and use of these organoids for experimentation was approved by the ethical committee at University Medical Center Utrecht (UMCU) (TcBio 14-008). Human stomach organoids, derived from normal corpus and pylorus, were from patients that underwent partial or total gastrectomy at the University Medical Centre Utrecht (UMCU) and were established as described19, 64, 65. Pancreas organoids were obtained from the healthy part of the pancreas of patients undergoing surgical resection of a tumour at the University Medical Centre Utrecht Hospital (UMC) and were established as described66, 67. The liver organoids were derived from freshly isolated normal liver tissue from a patient with metastatic CRC who presented at the UMC hospital (ethical approval code TCBio 14-007) and were established as described20, 68. For the performance of 3D cultures, Matrigel (BD Biosciences) was used and overlaid with a liquid medium consisting of DMEM/F12 advanced medium (Invitrogen), supplemented with additional factors as outlined below. 2% RSPO3-CM (produced via the r-PEX protein expression platform at U-Protein Express BV), WNT3A conditioned medium (50%, produced using stably transfected L cells in the presence of DMEM/F12 advanced medium supplemented with 10% FBS), and Wnt and Wnt/RSPO2 surrogates at different concentrations were added as indicated. Single-cell suspensions of normal human organoids were cultured in duplicate or triplicate in round-bottom 96-well plates to perform a cell viability test using Cell Titer-Glo 3D (Promega). In brief, organoids were trypsinized to single-cell suspension and plated in 100 μl medium in the presence of the different reagents. 3 μM IWP-2 was added to inhibit endogenous Wnt lipidation and secretion. After 12 days, 100 μl of Cell Titer-Glo 3D was added, plates were shaken for 5 min, incubated for an additional 25 min and centrifuged before luminescence measurement. All animal experiments were conducted in accordance with procedures approved by the IACUC at Stanford University. Experiments were not randomized, the investigators were not blinded, and all samples/data were included in the analysis. Group sample sizes were chosen based on (1) previous experiments, (2) performance of statistics analysis, and (3) logistical reasons with respect to full study size, to accommodate all groups. Adenoviruses (E1 and E3 deleted, replication deficient) were constructed to express scFv–DKK1c or scFv–DKK1c–RSPO2 with an N-terminal signal peptide and C-terminal 6×His-tag (Ad-scFv–DKK1c or Ad-scFv–DKK1c–RSPO2), respectively. Adenoviruses expressing mouse IgG2α Fc (Ad-Fc), human RSPO2–Fc fusion protein (Ad-RSPO2–Fc) and mouse WNT3A (Ad-Wnt3a) were constructed and described in the companion paper by Yan et al.26 The adenoviruses were cloned, purified by CsCl gradient, and titred as previously described69. Adult C57Bl/6J mice were purchased from Taconic Biosciences. Adult C57Bl/6J mice between 8–10 weeks old were injected intravenously with a single dose of adenovirus at between 1.2 × 107 p.f.u. to 6 × 108 p.f.u. per mouse in 0.1 ml PBS. Serum expression of Ad-scFv–DKK1c or Ad-scFv–DKK1c–RSPO2 were confirmed by immunoblotting using mouse anti-6×His (Abcam ab18184, 1:2,000) or rabbit anti-6×His (Abcam ab9108, 1:1,000), respectively. All experiments used n = 4 mice per group and repeated at least twice. qRT–PCR on liver samples were performed as following. Total cDNA was prepared from each liver sample using Direct-Zol RNA miniprep kit (Zymo Research) and iScript Reverse Transcription Supermix for RT-qPCR (BIO-RAD). Gene expression was analysed by -ΔΔC or fold change (2−ΔΔCt). Unpaired Student’s t-test (two tailed) was used to analyse statistical significance. Primers for mouse Axin2 and Cyp2f2 were previously published70. Additional primers used were listed as below: For the parabiosis experiment, age- and gender-matched C57Bl/6J mice were housed together for at least 2 weeks before surgery. At 2 days before surgery, the ‘donor’ mice were injected intravenously with a single dose of adenovirus at between 1.2 × 107 pfu to 6 × 108 pfu per mouse in 0.1 ml PBS and were separated from the ‘recipient’ mice until surgery. The parabiosis surgery was performed as described previously71. The establishment of shared circulation was confirmed at day 5 after surgery by presence of adenovirus-expressed proteins in the serum of both donors and recipients. Mouse livers were collected and fixed in 4% paraformaldehyde. 5 μm paraffin-embedded sections were stained with the following antibodies after citrate antigen retrieval and blocking with 10% normal goat serum: mouse anti-glutamine synthetase antibody (Millipore MAB302, 1:200), mouse anti-PCNA (BioLegend 307902, 1:200), and rabbit anti-HNF4α (Cell Signaling 3113S, 1:500). The immunostained tissue sections were analysed and images were captured on a Zeiss Axio-Imager Z1 with ApoTome attachment. Atomic structure factors and coordinates have been deposited to the Protein Data Bank (PDB) under accession numbers 5UN5 and 5UN6. All other data are available from the corresponding author upon reasonable request.


News Article | May 10, 2017
Site: www.scientificamerican.com

The origins of space and time are among the most mysterious and contentious topics in science. Our February 2017 article “Pop Goes the Universe” argues against the dominant idea that the early cosmos underwent an extremely rapid expansion called inflation. Its authors instead advocate for another scenario—that our universe began not with a bang but with a bounce from a previously contracting cosmos. In the letter below, a group of 33 physicists who study inflationary cosmology respond to that article. It is followed by a reply from the authors (an extended version of their reply can be found here). In “Pop Goes the Universe,” by Anna Ijjas, Paul J. Steinhardt and Abraham Loeb, the authors (hereafter “IS&L”) make the case for a bouncing cosmology, as was proposed by Steinhardt and others in 2001. They close by making the extraordinary claim that inflationary cosmology “cannot be evaluated using the scientific method” and go on to assert that some scientists who accept inflation have proposed “discarding one of [science’s] defining properties: empirical testability,” thereby “promoting the idea of some kind of nonempirical science.” We have no idea what scientists they are referring to. We disagree with a number of statements in their article, but in this letter, we will focus on our categorical disagreement with these statements about the testability of inflation. There is no disputing the fact that inflation has become the dominant paradigm in cosmology. Many scientists from around the world have been hard at work for years investigating models of cosmic inflation and comparing these predictions with empirical observations. According to the high-energy physics database INSPIRE, there are now more than 14,000 papers in the scientific literature, written by over 9,000 distinct scientists, that use the word “inflation” or “inflationary” in their titles or abstracts. By claiming that inflationary cosmology lies outside the scientific method, IS&L are dismissing the research of not only all the authors of this letter but also that of a substantial contingent of the scientific community. Moreover, as the work of several major, international collaborations has made clear, inflation is not only testable, but it has been subjected to a significant number of tests and so far has passed every one. Inflation is not a unique theory but rather a class of models based on similar principles. Of course, nobody believes that all these models are correct, so the relevant question is whether there exists at least one model of inflation that seems well motivated, in terms of the underlying particle physics assumptions, and that correctly describes the measurable properties of our universe. This is very similar to the early steps in the development of the Standard Model of particle physics, when a variety of quantum field theory models were explored in search of one that fit all the experiments. Although there is in principle a wide space of inflationary models to examine, there is a very simple class of inflationary models (technically, “single-field slow-roll” models) that all give very similar predictions for most observable quantities—predictions that were clearly enunciated decades ago. These “standard” inflationary models form a well-defined class that has been studied extensively. (IS&L have expressed strong opinions about what they consider to be the simplest models within this class, but simplicity is subjective, and we see no reason to restrict attention to such a narrow subclass.) Some of the standard inflationary models have now been ruled out by precise empirical data, and this is part of the desirable process of using observation to thin out the set of viable models. But many models in this class continue to be very successful empirically. The standard inflationary models predict that the universe should have a critical mass density (that is, it should be geometrically flat), and they also predict the statistical properties of the faint ripples that we detect in the cosmic microwave background (CMB). First, the ripples should be nearly “scale-invariant,” meaning that they have nearly the same intensity at all angular scales. Second, the ripples should be “adiabatic,” meaning that the perturbations are the same in all components: the ordinary matter, radiation and dark matter all fluctuate together. Third, they should be “Gaussian,” which is a statement about the statistical patterns of relatively bright and dark regions. Fourth and finally, the models also make predictions for the patterns of polarization in the CMB, which can be divided into two classes, called E-modes and B-modes. The predictions for the E-modes are very similar for all standard inflationary models, whereas the levels of B-modes, which are a measure of gravitational radiation in the early universe, vary significantly within the class of standard models. The remarkable fact is that, starting with the results of the Cosmic Background Explorer (COBE) satellite in 1992, numerous experiments have confirmed that these predictions (along with several others too technical to discuss here) accurately describe our universe. The average mass density of the universe has now been measured to an accuracy of about half of a percent, and it agrees perfectly with the prediction of inflation. (When inflation was first proposed, the average mass density was uncertain by at least a factor of three, so this is an impressive success.) The ripples of the CMB have been measured carefully by two more satellite experiments, the Wilkinson Microwave Anisotropy Probe (WMAP) and the Planck satellite, as well as many ground- and balloon-based experiments—all confirming that the primordial fluctuations are indeed nearly scale-invariant and very accurately adiabatic and Gaussian, precisely as predicted (ahead of time) by standard models of inflation. The B-modes of polarization have not yet been seen, which is consistent with many, though not all, of the standard models, and the E-modes are found to agree with the predictions. In 2016 the Planck satellite team (a collaboration of about 260 authors) summarized its conclusions by saying that “the Planck results offer powerful evidence in favour of simple inflationary models.” So if inflation is untestable, as IS&L would have us believe, why have there been so many tests of it and with such remarkable success? While the successes of inflationary models are unmistakable, IS&L nonetheless make the claim that inflation is untestable. (We are bewildered by IS&L’s assertion that the dramatic observational successes of inflation should be discounted while they accuse the advocates of inflation of abandoning empirical science!) They contend, for example, that inflation is untestable because its predictions can be changed by varying the shape of the inflationary energy density curve or the initial conditions. But the testability of a theory in no way requires that all its predictions be independent of the choice of parameters. If such parameter independence were required, then we would also have to question the status of the Standard Model, with its empirically determined particle content and 19 or more empirically determined parameters. An important point is that standard inflationary models could have failed any of the empirical tests described above, but they did not. IS&L write about how “a failing theory gets increasingly immunized against experiment by attempts to patch it,” insinuating that this has something to do with inflation. But despite IS&L’s rhetoric, it is standard practice in empirical science to modify a theory as new data come to light, as, for example, the Standard Model has been modified to account for newly discovered quarks and leptons. For inflationary cosmology, meanwhile, there has so far been no need to go beyond the class of standard inflationary models. IS&L also assert that inflation is untestable because it leads to eternal inflation and a multiverse. Yet although the possibility of a multiverse is an active area of study, this possibility in no way interferes with the empirical testability of inflation. If the multiverse picture is valid, then the Standard Model would be properly understood as a description of the physics in our visible universe, and similarly the models of inflation that are being refined by current observations would describe the ways inflation can happen in our particular part of the universe. Both theories would remain squarely within the domain of empirical science. Scientists would still be able to compare newly obtained data—from astrophysical observations and particle physics experiments—with precise, quantitative predictions of specific inflationary and particle physics models. Note that this issue is separate from the loftier goal of developing a theoretical framework that can predict, without the use of observational data, the specific models of particle physics and inflation that should be expected to describe our visible universe. Like any scientific theory, inflation need not address all conceivable questions. Inflationary models, like all scientific theories, rest on a set of assumptions, and to understand those assumptions we might need to appeal to some deeper theory. This, however, does not undermine the success of inflationary models. The situation is similar to the standard hot big bang cosmology: the fact that it left several questions unresolved, such as the near-critical mass density and the origin of structure (which are solved elegantly by inflation), does not undermine its many successful predictions, including its prediction of the relative abundances of light chemical elements. The fact that our knowledge of the universe is still incomplete is absolutely no reason to ignore the impressive empirical success of the standard inflationary models. During the more than 35 years of its existence, inflationary theory has gradually become the main cosmological paradigm describing the early stages of the evolution of the universe and the formation of its large-scale structure. No one claims that inflation has become certain; scientific theories don’t get proved the way mathematical theorems do, but as time passes, the successful ones become better and better established by improved experimental tests and theoretical advances. This has happened with inflation. Progress continues, supported by the enthusiastic efforts of many scientists who have chosen to participate in this vibrant branch of cosmology. Empirical science is alive and well! »Click here to jump to the authors’ reply Alan H. Guth             Victor F. Weisskopf Professor of Physics, Massachusetts Institute of Technology             http://web.mit.edu/physics/people/faculty/guth_alan.html David I. Kaiser             Germeshausen Professor of the History of Science and Professor of Physics, Massachusetts Institute of Technology             http://web.mit.edu/physics/people/faculty/kaiser_david.html Andrei D. Linde             Harald Trap Friis Professor of Physics, Stanford University             https://physics.stanford.edu/people/faculty/andrei-linde Yasunori Nomura             Professor of Physics and Director, Berkeley Center for Theoretical Physics, University of California, Berkeley             http://physics.berkeley.edu/people/faculty/yasunori-nomura Charles L. Bennett             Bloomberg Distinguished Professor and Alumni Centennial Professor of Physics and Astronomy, Johns Hopkins University             Principal Investigator, Wilkinson Microwave Anisotropy Probe (WMAP) mission             Deputy Principal Investigator and Science Working Group member, Cosmic Background Explorer (COBE) mission             http://physics-astronomy.jhu.edu/directory/charles-l-bennett/ J. Richard Bond             University Professor, University of Toronto and Director, Canadian Institute for Advanced Research Cosmology and Gravity Program, Canadian Institute for Theoretical Astrophysics             Member of the Planck collaboration             http://www.cita.utoronto.ca/~bond/ François Bouchet             Director of Research, Institut d’Astrophysique de Paris, CNRS and Sorbonne Université-UPMC             Deputy Principal Investigator, Planck satellite HFI (High Frequency Instrument) Consortium and Member, Planck Science Team             http://savoirs.ens.fr/conferencier.php?id=145 Sean Carroll             Research Professor of Physics, California Institute of Technology             http://www.astro.caltech.edu/people/faculty/Sean_Carroll.html George Efstathiou             Professor of Astrophysics, Kavli Institute for Cosmology, University of Cambridge             Member, Planck Science Team             http://www.ast.cam.ac.uk/~gpe/ Stephen Hawking             Lucasian Professor of Mathematics (Emeritus) and Dennis Stanton Avery and Sally Tsui Wong-Avery Director of Research, Department of Applied Mathematics and Theoretical Physics, University of Cambridge             http://www.damtp.cam.ac.uk/people/s.w.hawking/ Renata Kallosh             Professor of Physics, Stanford University             https://physics.stanford.edu/people/faculty/renata-kallosh Eiichiro Komatsu             Director of the Department of Physical Cosmology, Max-Planck-Institute für Astrophysik, Garching             Member, Wilkinson Microwave Anisotropy Probe (WMAP) collaboration             http://wwwmpa.mpa-garching.mpg.de/~komatsu/ Lawrence Krauss             Foundation Professor in the School of Earth and Space Exploration and Department of Physics, and Director, The Origins Project at Arizona State University             http://krauss.faculty.asu.edu David H. Lyth             Professor of Physics (Emeritus), Lancaster University             http://www.lancaster.ac.uk/physics/about-us/people/david-lyth Juan Maldacena             Carl P. Feinberg Professor of Physics, Institute for Advanced Study             https://www.sns.ias.edu/malda John C. Mather             Senior Astrophysicist and Goddard Fellow, NASA Goddard Space Flight Center and recipient of the Nobel Prize in Physics (2006)             Project Scientist, Cosmic Background Explorer (COBE) mission and             Senior Project Scientist, James Webb Space Telescope             https://science.gsfc.nasa.gov/sed/bio/john.c.mather Hiranya Peiris             Professor of Astrophysics, University College London and Director, Oskar Klein Centre for Cosmoparticle Physics, Stockholm             Member, Wilkinson Microwave Anisotropy Probe (WMAP) collaboration and Planck collaboration             http://zuserver2.star.ucl.ac.uk/~hiranya/ Malcolm Perry             Professor of Theoretical Physics, University of Cambridge             http://www.damtp.cam.ac.uk/people/m.j.perry/ Lisa Randall             Frank B. Baird, Jr., Professor of Science, Department of Physics, Harvard University             https://www.physics.harvard.edu/people/facpages/randall Martin Rees             Astronomer Royal of Great Britain, former President of the Royal Society of London, and Professor (Emeritus) of Cosmology and Astrophysics, University of Cambridge             http://www.ast.cam.ac.uk/~mjr/ Misao Sasaki             Professor, Yukawa Institute for Theoretical Physics, Kyoto University             http://www2.yukawa.kyoto-u.ac.jp/~misao.sasaki/ Leonardo Senatore             Associate Professor of Physics, Stanford University             https://physics.stanford.edu/people/faculty/leonardo-senatore Eva Silverstein             Professor of Physics, Stanford University             https://physics.stanford.edu/people/faculty/eva-silverstein George F. Smoot III             Professor of Physics (Emeritus), Founding Director, Berkeley Center for Cosmological Physics, and recipient of the Nobel Prize in Physics (2006)             Principal Investigator, Cosmic Background Explorer (COBE) mission             http://physics.berkeley.edu/people/faculty/george-smoot-iii Alexei Starobinsky             Principal Researcher, Landau Institute for Theoretical Physics, Moscow             http://www.itp.ac.ru/en/persons/starobinsky-aleksei-aleksandrovich/ Leonard Susskind             Felix Bloch Professor of Physics and Wells Family Director, Stanford Institute for Theoretical Physics, Stanford University             https://physics.stanford.edu/people/faculty/leonard-susskind Michael S. Turner             Bruce. V. Rauner Distinguished Service Professor, Department of Astronomy and Astrophysics and Department of Physics, University of Chicago             https://astro.uchicago.edu/people/michael-s-turner.php Alexander Vilenkin             L. and J. Bernstein Professor of Evolutionary Science and Director, Institute of Cosmology, Tufts University             http://cosmos2.phy.tufts.edu/vilenkin.html Steven Weinberg             Jack S. Josey-Welch Foundation Chair and Regental Professor and Director, Theory Research Group, Department of Physics, University of Texas at Austin, and recipient of the Nobel Prize in Physics (1979)             https://web2.ph.utexas.edu/~weintech/weinberg.html Rainer Weiss                         Professor of Physics (Emeritus), Massachusetts Institute of Technology             Chair, Science Working Group, Cosmic Background Explorer (COBE) mission             Co-Founder, Laser Interferometric Gravitational-wave Observatory (LIGO)             http://web.mit.edu/physics/people/faculty/weiss_rainer.html Frank Wilczek                      Herman Feshbach Professor of Physics, Massachusetts Institute of Technology, and recipient of the Nobel Prize in Physics (2004)             http://web.mit.edu/physics/people/faculty/wilczek_frank.html Edward Witten                      Charles Simonyi Professor of Physics, Institute for Advanced Study and recipient of the Fields Medal (1990)             https://www.sns.ias.edu/witten Matias Zaldarriaga               Professor of Astrophysics, Institute for Advanced Study             https://www.sns.ias.edu/matiasz THE AUTHORS REPLY: We have great respect for the scientists who signed the rebuttal to our article, but we are disappointed by their response, which misses our key point: the differences between the inflationary theory once thought to be possible and the theory as understood today. The claim that inflation has been confirmed refers to the outdated theory before we understood its fundamental problems. We firmly believe that in a healthy scientific community, respectful disagreement is possible and hence reject the suggestion that by pointing out problems, we are discarding the work of all of those who developed the theory of inflation and enabled precise measurements of the universe. Historically, the thinking about inflation was based on a series of misunderstandings. It was not understood that the outcome of inflation is highly sensitive to initial conditions. And it was not understood that inflation generically leads to eternal inflation and, consequently, a multiverse—an infinite diversity of outcomes. Papers claiming that inflation predicts this or that ignore these problems. Our point is that we should be talking about the contemporary version of inflation, warts and all, not some defunct relic. Logically, if the outcome of inflation is highly sensitive to initial conditions that are not yet understood, as the respondents concede, the outcome cannot be determined. And if inflation produces a multiverse in which, to quote a previous statement from one of the responding authors (Guth), “anything that can happen will happen”—it makes no sense whatsoever to talk about predictions. Unlike the Standard Model, even after fixing all the parameters, any inflationary model gives an infinite diversity of outcomes with none preferred over any other. This makes inflation immune from any observational test. For more details, see our 2014 paper “Inflationary Schism” (preprint available at https://arxiv.org/abs/1402.6980). We are three independent thinkers representing different generations of scientists. Our article was not intended to revisit old debates but to discuss the implications of recent observations and to point out unresolved issues that present opportunities for a new generation of young cosmologists to make a lasting impact. We hope readers will go back and review our article’s concluding paragraphs. We advocated against invoking authority and for open recognition of the shortcomings of current concepts, a reinvigorated effort to resolve these problems and an open-minded exploration of diverse ideas that avoid them altogether. We stand by these principles.

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