ARTICLE WATCH
Article Watch: September 2016 Clive A. Slaughter This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, AU-UGA Medical Partnership, 1425 Prince Ave., Athens, GA 30606, USA. Tel: (706) 713-2216; Fax: (706) 713-2221; E-mail: [email protected], or to any member of the editorial board. Article summaries reflect the reviewer’s opinions and not necessarily those of the association.
METABOLOMICS
Cahill J F, Kertesz V, Weiskittel T M, Vavrek M, Freddo C, Van Berkel G J. Online, absolute quantitation of propranolol from spatially distinct 20- and 40-mm dissections of brain, liver, and kidney thin tissue sections by laser microdissection–liquid vortex capture–mass spectrometry. Analytical Chemistry 88;2016: 6026–6034. This paper describes methodology for mass spectrometric absolute quantification of a drug molecule (propranolol) and its metabolites in tissue sections that provides high spatial resolution. A 10 mm-thick tissue section collected onto a polyethylene naphthalate membrane glass slide is mounted in a commercial laser capture microdissection system. The laser cuts from the tissue-section, squareshaped samples, measuring 20 3 20 mm or 40 3 40 mm. An additional laser pulse aimed at the cut sample causes it to fall into a stream of flowing solvent that transports it to the electrospray (ESI) probe of an online mass spectrometer. The drug is extracted from the tissue dissolving in the solvent stream. The ratio of propranolol signal strength to the signal strength of a fixed concentration of propranolol-d7 spiked into the solvent is measured and compared with a standard curve constructed with known quantities of propranolol. Spatial resolution in this study was limited only by the sensitivity of detection of the analyte: sample sizes as small as 5–10 mm diameter are reported to be achievable with the dissection system. The measurements compare well with values obtained using a tissue punch and HPLC/ESI/mass spectrometry (MS)/MS. The methodology requires no addition of matrix, as would be needed for matrix-assisted laser desorption/ionization (MALDI) MS. The system can, in principle, be used for measuring multiple analytes in a lipidomics-type approach.
doi: 10.7171/jbt.16-2703-005
Journal of Biomolecular Techniques 27:119–124 © 2016 ABRF
CARBOHYDRATES AND GLYCOCONJUGATES
Song X, Ju H, Lasanajak Y, Kudelka M R, Smith D F, Cummings R D. Oxidative release of natural glycans for functional glycomics. Nature Methods 13;2016:528–534. Glycans are generally released from glycoconjugates for the purposes of structural and functional analysis by treatment with expensive enzymes or corrosive or toxic chemicals. Most such procedures are suitable for only small amounts of starting material and are not readily amenable to scaling up. Song et al. note that the use of commercial bleach for sterilization exploits the strong oxidative power of its active ingredient, sodium hypochlorite (NaClO), toward proteins in microorganisms. In the present paper, they therefore explore the use of NaClO for oxidative release of glycans from glycoconjugates as a potentially simple, inexpensive procedure suitable for use on a preparative scale. They show that treatment of various glycoproteins with 1% NaClO degrades the protein moiety within minutes. Free N-glycans are released as glycosamines, which spontaneously convert to free reducing glycans in aqueous solution at room temperature. Otherwise-labile sialic acid residues are well preserved. The glycans can react with amines, allowing addition of fluorescent tags that facilitate chromatographic analysis and purification. The authors demonstrate release of N-glycans from sources that include human saliva and chicken egg whites and yolks and characterize these glycans by MS and 1H NMR. They also show that the fractionated N-glycans immobilized on microarrays are able to bind specific plant lectins. The authors go on to demonstrate that NaClO releases O-glycan from mucins and fetuin, although the greater stability of O-glycosidic linkages requires a higher concentration of NaClO. The glycans released are found to be linked at the reducing end to glycolic acid (presumably derived from serine) or lactic acid (presumably derived from threonine). Finally, the authors show that NaClO releases glycans from glycosphingolipids as glycan nitriles. The nitrile can also be used for fluorescent tagging. Oxidative
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release of glycans with NaClO is anticipated to facilitate the archiving and characterization of diverse glycans from animal and human sources.
PROTEOMICS
Abelin J G, Patel J, Lu X, Feeney C M, Fagbami L, Creech A L, Hu R, Lam D, Davison D, Pino L, Qiao J W, Kuhn E, Officer A, Li J, Abbatiello S, Subramanian A, Sidman R, Snyder E, Carr S A, Jaffe J D. Reduced-representation phosphosignatures measured by quantitative targeted MS capture cellular states and enable large-scale comparison of drug-induced phenotypes. Molecular & Cellular Proteomics 15;2016:1622–1641. Prominent among cellular responses to perturbation by drugs, stressors, hormones, interactions with extracellular matrix, and contact with neighboring cells are responses mediated by changes in signaling pathways comprised of protein kinases. These pathways affect the phosphorylation state of very large numbers of phosphate acceptor sites on proteins. For example, .70,000 sites of phosphorylation on serine and threonine residues are known in the human proteome. Such a number is far too large to monitor in each of the thousands of samples that one would like to analyze in studies of cell physiology, but only a few hundred protein kinases determine the occupancy of all of these phosphorylation states. This suggests the possibility that occupancy of many of the sites might be coordinately regulated and that the monitoring of a small subset of them might provide an adequate description of the various responses to perturbation that cells can mount. Abelin et al. here investigate this possibility. They study the phosphorylation state of sites in 3 cell lines treated with 26 different bioactive small molecules, cluster the observed responses, and pick representative phosphopeptides from each cluster for monitoring. They develop a targeted parallel reaction monitoring assay to measure 96 of these phosphopeptides. Sample processing is fully automated, so sample throughput is maximized and technical variance minimized. The procedure allowed 95% of the phosphopeptides to be detected in ;200 samples. The results reproduce cell-state classifications previously formulated on the basis of much more extensive datasets acquired in discovery experiments. The performance of the assay is then verified by measuring the same phosphopeptides in new samples generated under new treatment conditions, cell types, and time points. The assay is shown to be sensitive to perturbations in a diverse set of common signaling pathways. This reduced representation of phosphoprofiling assay is believed to be suitable for large-scale 120
phosphoproteome studies involving longitudinal comparisons among thousands of samples and, therefore, is hoped to contribute to an understanding of cellular responses to diverse stimuli. Wilson R E, Groskreutz S R, Weber S G. Improving the sensitivity, resolution, and peak capacity of gradient elution in capillary liquid chromatography with large-volume injections by using temperatureassisted on-column solute focusing. Analytical Chemistry 88;2016:5112–5121. Capillary columns are in widespread use for separating peptides by HPLC in online mass spectrometric analysis. For convenience of sample preparation, sample volumes are often large compared with the volume of the capillary separation bed. This may lead to broadening of peaks for analytes that elute early in a gradient separation. Short, hydrophilic peptides exhibit such broadening during reverse-phase separation. The authors of the present paper offer a remedy for this problem. Because analytes are more strongly retained at lower column temperature, the column is maintained at subambient temperature (27.5°C) during sample loading. The column temperature is then increased quickly to one suitable for chromatographic separation (65°C) before starting gradient elution. The process is fully automated and requires special hardware. It provides significant improvement in detection sensitivity, peak resolution, and peak capacity for small peptides. Shao S, Guo T, Gross V, Lazarev A, Koh C C, Gillessen S, Joerger M, Jochum W, Aebersold R. Reproducible tissue homogenization and protein extraction for quantitative proteomics using MicroPestle-assisted pressure-cycling technology. Journal of Proteome Research 15;2016:1821–1829. Reproducible and efficient extraction of proteins from the small amounts of tissue in clinical biopsy samples is challenging. Automated pressure cycling technology has been developed to meet this need. Samples are subjected to alternating high and low pressure in a Barocycler device for disruption without mechanical shearing. Shao et al. here describe improvements in this technology. A disposable, close-fitting Teflon pestle is inserted into a microtube containing the sample and buffer. During pressure pulses, the tube walls collapse around the incompressible pestle, producing an homogenizer-like effect. The methodology is demonstrated in proteomic studies of mouse liver, heart, brain, and human kidney. JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 27, ISSUE 3, SEPTEMBER 2016
SLAUGHTER / ARTICLE WATCH
FUNCTIONAL GENOMICS AND PROTEOMICS
Sadhu M J, Bloom J S, Day L, Kruglyak L. CRISPRdirected mitotic recombination enables genetic mapping without crosses. Science 352;2016: 1113–1116. Linkage and association studies are conducted by associating a phenotype with genetic markers that define a chromosome region. The identification of which specific gene within such a region of interest is responsible for a given phenotype, however, is much more difficult, as it relies on rare recombination events within the region to distinguish the effects of the included genes. Sadhu et al. here use clustered, regularly interspaced, short palindromic repeats (CRISPR) to generate recombination events targeted to predetermined locations within genomic regions of interest for the purposes of fine (high-resolution) genetic mapping. They use the CRISPR-CRISPR associated protein 9 (Cas9) system to induce double-stranded breaks at chosen sites in a chromosome of a diploid cell undergoing mitosis. The homologous chromosome is left intact to serve as a template for repair by homologous recombination, a process that results in loss of heterozygosity (gene conversion). The authors show that CRISPRinduced recombination occurs with high efficiency and with few off-target effects. The authors test their system by generating a recombination panel in yeast cells to map a phenotype (manganese sensitivity) to a polymorphism in a particular transporter (Pmr1). Such trait mapping with targeted loss-of-heterozygosity panels is anticipated to become a standard method for localizing the genetic basis of phenotypic variation. Evers B, Jastrzebski K, Heijmans J P M, Grernrum W, Beijersbergen R L, Bernards R. CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes. Nature Biotechnology 34;2016:631–633. Morgens D W, Deans R M, Li A, Bassik M C. Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes. Nature Biotechnology 34; 2016:634–636. Two groups compare the effectiveness of RNA interference (RNAi) and CRISPR methodology in screening for essential genes in mammalian cultured cells. A similar approach is used by both groups. They use pooled libraries of short hairpin RNA (shRNA) or CRISPR single-guide RNA to look for dropout of gold-standard essential and nonessential genes by high-throughput sequencing. In this JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 27, ISSUE 3, SEPTEMBER 2016
way, they seek to determine the sensitivity and specificity of each methodology. Evers et al. find that CRISPR performs better than RNAi, whereas Morgens et al. find that the 2 methods perform similarly. The 2 groups do conduct testing with RNA libraries of different complexity: for example, the shRNA library of Evers et al. has 4.8 RNA reagents per gene, whereas that of Morgens et al. has 25. Such a difference could explain the superior results observed by Morgens et al. with RNAi. Such differences notwithstanding, the data suggest that the choice of genes for targeting and the choice of RNA screening libraries might affect as big of an influence on outcome as the methodological approach chosen. Of special interest is that both groups observed less than the expected overlap in the list of essential genes detected by RNAi and CRISPR. The mode of action of the 2 methods appears to select for identification of somewhat different sets of genes. The basis for these differences remains to be determined and will be of particular interest to investigators using these methods for loss-of-function and other kinds of assays. Morgens et al. additionally provide computational methods for combining the results of RNAi and CRISPR screens. IMAGING
Morisaki T, Lyon K, DeLuca K F, DeLuca J G, English B P, Zhang Z, Lavis L D, Grimm J B, Viswanathan S, Looger L L, Lionnet T, Stasevich T J. Real-time quantification of single RNA translation dynamics in living cells. Science 352;2016: 1425–1429. Wang C, Han B, Zhou R, Zhuang X. Real-time imaging of translation on single mRNA transcripts in live cells. Cell 165;2016:990–1001. Wu B, Eliscovich C, Yoon Y J, Singer R H. Translation dynamics of single mRNAs in live cells and neurons. Science 352;2016:1430–1435. Yan X, Hoek Tim A, Vale Ronald D, Tanenbaum Marvin E. Dynamics of translation of single mRNA molecules in vivo. Cell 165;2016:976–989. Four groups have explored methods for imaging nascent polypeptides during synthesis on single mRNA molecules within living cells. To visualize polypeptides as they emerge from the ribosome, all 4 groups incorporate tandem repeats of a polypeptide epitope that rapidly bind pre-existing fluorescent antibodies following ribosomal assembly and use fluorescent RNA-binding proteins to detect colocalized, single mRNA molecules. The imaging studies enabled by these techniques reveal initiation rates, 121
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elongation speeds, and ribosome spacing on polysomes in a cellular context. By focusing on individual mRNA molecules, they also reveal spatial and temporal variation in translational activity, gene-specific effects, and characteristics of intracellular polysome mobility. The present studies used exogenous message constructs, but similar approaches based on genome editing could be used to reveal details of tissue-specific translational control during development. Ngo J T, Adams S R, Deerinck T J, Boassa D, Rodriguez-Rivera F, Palida S F, Bertozzi C R, Ellisman M H, Tsien R Y. Click-EM for imaging metabolically tagged nonprotein biomolecules. Nature Chemical Biology 12;2016:459–465. Ngo et al. describe a technique for electron microscope (EM) imaging of selected nonprotein biomolecules, such as glycans, nucleic acids and lipids, at nanometer resolution. They adopt a metabolic labeling approach in which metabolic analogs of monosaccharides, nucleotides, or fatty acids containing a bio-orthogonal functional group are provided to cells for incorporation into biomolecules by endogenous biosynthetic processes. The functional group is then targeted for attachment of a fluorescent dye by click chemistry. The dye generates singlet oxygen upon photolysis, and the singlet oxygen mediates local polymerization of diaminobenzidine to form a polymeric precipitate that can be stained with osmium tetroxide for visualization by EM. The dye-labeled biomolecules can also be imaged by fluorescence microscopy for correlation with the EM images. The authors use this procedure to image DNA, RNA, and lipids in cultured cells and neurons and to track peptidoglycan synthesis in a grampositive bacterium. The methodology does not involve permeabilization of cells, which might disrupt ultrastructural features. The methodology also introduces reagents required for EM contrast only after fixing the cells. It enables high resolution by ensuring that the deposition of contrast materials occurs within nanometers of the structures of interest. The authors envision application of the technique to studying structures that are difficult to resolve by light microscopy, such as axon terminals, mitochondria, synaptic vesicles, and autophagosomes. Wu Q, Comi T J, Li B, Rubakhin S S, Sweedler J V. On-tissue derivatization via electrospray deposition for matrix-assisted laser desorption/ionization mass spectrometry imaging of endogenous fatty acids in rat brain tissues. Analytical Chemistry 88;2016:5988–5995. Imaging by MALDI is of limited efficacy for molecules that are small enough to incur interference by matrix ions, 122
molecules of low abundance, and molecules that ionize poorly. Wu et al. here describe a method for MALDI imaging of free fatty acids, a class of biomolecules that includes species of interest for the synthesis of signaling molecules and lipid metabolism but that ionizes poorly in the positive ion mode. They enhance the detectability of these molecules by specific derivatization with 2-picolylamine in the presence of 2,2-dipyridyl disulfide and triphenylphosphine. This adds a pyridyl moiety to the carboxyl group via amide linkage, promoting formation of positive ions. The reaction occurs rapidly over a broad temperature range, making it suitable for derivatization on tissue slices. Importantly, the derivatization reagent is applied by electrospray deposition. Electrospray produces charged, nanoscale droplets that promote extraction and derivatization of target fatty acids but minimizes their delocalization. With the use of this procedure, the authors visualized 6 fatty acids in selected regions of the rat brain with effective spatial resolution of 20 mm and were able to distinguish fatty acid distribution in adjacent cell layers of the hippocampus. They expect that electrospray-assisted derivatization will prove useful for imaging further classes of analyte. CELL BIOLOGY AND TISSUE ENGINEERING
Shahbazi M N, Jedrusik A, Vuoristo S, Recher G, Hupalowska A, Bolton V, Fogarty N M E, Campbell A, Devito L G, Ilic D, Khalaf Y, Niakan K K, Fishel S, Zernicka-Goetz M. Self-organization of the human embryo in the absence of maternal tissues. Nature Cell Biology 18;2016:700–708. Deglincerti A, Croft G F, Pietila L N, ZernickaGoetz M, Siggia E D, Brivanlou A H. Selforganization of the in vitro attached human embryo. Nature 533;2016:251–254. During human embryonic development, the fertilized egg divides to form a free-floating blastocyst consisting of 3 cell lineages: the epiblast, hypoblast, and trophectoderm. Following blastocyst implantation into the uterine wall at d 5, these lineages produce the fetus, extraembryonic yolk sac, and placenta, respectively. Study of the remodeling that occurs in human embryos at implantation has hitherto been impeded by lack of suitable in vitro culture conditions. (The value of experiments with nonhuman animals is limited by significant differences between species in timing and control of the events.) Two groups now describe culture conditions that permit in vitro observation throughout the implantation stages. They show that blastocysts, derived from supernumerary fertilized eggs, attach to the culture dish. The attached blastocysts exhibit key transitions, including JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 27, ISSUE 3, SEPTEMBER 2016
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segregation of embryonic and extraembryonic cell lineages, epiblast polarization, formation of the proamniotic cavity and bilaminar disk, appearance of a presumptive yolk sac, and differentiation of the trophoblast into the cytotrophoblast and syncytiotrophoblast. The occurrence of these transitions in the absence of maternal tissues highlights the self-organizing capability of the human embryo. The improved technical capabilities described in these papers are expected to help understand the signaling processes that mediate normal implantation and its accompanying morphogenetic events and to gain insight into implantation failure, which is a major cause of early pregnancy loss. Both groups terminated their experiments at d 14 postfertilization (before formation of the primitive streak and the embryo germ layers) in compliance with long-standing international policy adopted to permit embryo research within ethical guidelines. It is likely that the present technical advances will prompt re-evaluation of this policy. (For analysis of policy and ethical considerations, see Hyun I, Wilkerson A, Johnson J. Nature 533;2016:170–171.) Hyslop L A, Blakeley P, Craven L, Richardson J, Fogarty N M E, Fragouli E, Lamb M, Wamaitha S E, Prathalingam N, Zhang Q, O’Keefe H, Takeda Y, Arizzi L, Alfarawati S, Tuppen H A, Irving L, Kalleas D, Choudhary M, Wells D, Murdoch A P, Turnbull D M, Niakan K K, Herbert M. Toward clinical application of pronuclear transfer to prevent mitochondrial DNA disease. Nature 534;2016:383–386. Hyslop et al. report preclinical studies of reproductive techniques to reduce the risk of human disease resulting from mutation in mitochondrial DNA. The method involves transplanting nuclei from oocytes with affected mitochondria to enucleated donor oocytes free of abnormalities in mitochondrial DNA. Initial studies indicated that nuclear transplantation between human oocytes that are arrested in meiosis division II results in a high incidence of abnormal fertilization. An alternative approach proves to be more successful. It involves transplanting the maternal and paternal genomes, separately packaged in discrete pronuclei, after in vitro fertilization. The authors find that this transplantation procedure gives best results when performed shortly after the completion of the second meiotic division, rather than shortly before the first mitotic division that produces a 2-cell embryo. This method results in improved survival and produces blastocysts of good morphologic quality. No effect on aneuploidy or gene expression is detected, and carryover of mitochondrial DNA is low. These preliminary results encourage further exploration of the technology JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 27, ISSUE 3, SEPTEMBER 2016
for reducing the risk of mitochondrial disease in the children of affected mothers. Treutlein B, Lee Q Y, Camp J G, Mall M, Koh W, Shariati S A M, Sim S, Neff N F, Skotheim J M, Wernig M, Quake S R. Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq. Nature 534;2016:391–395. Mouse embryonic fibroblasts can be directly reprogrammed to form induced neuronal cells by overexpression of 3 transcription factors: Ascl1, Brn2, and Myt1l. Transdifferentiation occurs within 2–3 wk in culture at an efficiency of up to 20%. Ascl1 alone also mediates transdifferentiation, but with lower efficiency. The present paper shows how single-cell RNA sequencing (RNA-Seq) can be used to analyze the molecular pathways by which transdifferentiation occurs and to identify processes that limit its efficiency. To reconstruct the progress of cells toward the neuronal phenotype under Ascl1 overexpression, the authors characterize the transcriptome of a total of 405 individual cells representing 0, 2, 5, and 22 d in culture. They find that the initial response, in terms of Ascl1 expression and silencing of fibroblast-associated genes, is relatively homogeneous among fibroblasts, with no evidence for variation in susceptibility to transdifferentiation. However, later, they observe cells that show Ascl1 silencing and emergence of a competing differentiation program that activates muscle-like (myogenic) genes, even though these cells turn on the neuronal marker, Tau. Overexpression of Brn2 and Myt1l, in addition to Ascl1, apparently suppresses the competing pathway. This study provides methodology by which the molecular processes responsible for reprogramming and differentiation stability may be studied. It also indicates the importance of using more than 1 marker (such as Tau) for identifying a differentiated state and the potential importance of using multiple transcription factors for directing reprogramming. MICROBIOME
Browne H P, Forster S C, Anonye B O, Kumar N, Neville B A, Stares M D, Goulding D, Lawley T D. Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation. Nature 533;2016:543–546. The bacterial assemblage that inhabits our gastrointestinal (GI) tract comprises complex ecological communities known as microbiota. Microbiota typically contain 100–1000 bacterial species. The recent increase in our knowledge of microbiota species diversity has relied heavily 123
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on methodology involving metagenomic sequencing that does not require the bacteria to be cultured. Indeed, it is generally believed that the majority of our commensal GI bacteria is recalcitrant to culturing. Browne et al. here demonstrate how a substantial majority of these bacteria is, in fact, amenable to culturing. The authors homogenize fecal samples from healthy human donors, serially dilute the homogenate, and inoculate it onto agar plates containing a complex, broad-range bacteriologic medium. Single colonies are then streaked to purity. All of these manipulations are conducted under anaerobic conditions, as GI bacteria are typically very sensitive to ambient oxygen. The pure colonies may then be characterized by genomic sequencing. Comparison of the sequences with the results of metagenomic sequencing of the original unfractionated feces indicates that 74% of the computationally derived metagenomic species in the unfractionated samples are represented in the pure cultures. The authors proceed to isolate, sequence, and archive 137 bacterial species in this way, including 45 presumptively novel species, 20 novel genera, and 2 novel families. Because spores of the pathogen Clostridium difficile are resistant to ethanol—a commonly used disinfectant—the authors use their system to characterize spore-forming bacteria identified as such by their ethanol resistance. The results indicate that 50–60% of the bacterial genera in healthy individuals produce spores. By sequencing, the authors define 66 conserved genes linked to the ethanol-resistant phenotype that may be used to predict sporulation capability in new bacterial isolates. The finding also helps to explain transmission of bacteria from 1 person to another. The capability to culture and sequence the genomes of substantial numbers of bacterial species from the human GI tract is expected to support important advances in knowledge of our commensal partners.
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POLICY
Stappenbeck T S, Virgin H W. Accounting for reciprocal host–microbiome interactions in experimental science. Nature 534;2016:191–199. It is increasingly apparent that variation in microbiota can affect the incidence and course of diseases, the physiologic processes on which diseases depend, and the results of treatment. Whereas the effects of GI microbiota on the intestinal epithelium are generally appreciated, awareness of the effects on other organ systems of commensals, located either in the GI tract or elsewhere, is weaker. For example, apparently heritable characteristics might be mediated by vertical transmission of microbes, as well as by transmission of host genes, and effects of age, circadian changes, and exposure to pathogens might all be strongly affected by commensal microbes. The effects of variables related to microbiota may lead to erroneous interpretation of experimental results if experimental design fails to control for them and may create confusion as a result of unreproducible findings. The authors of the present perspective suggest features of experimental design to mitigate such risks. For example, investigators using mouse models are advised to compare phenotypes of test and control animal groups that have been raised as littermates rather than to use controls purchased from a remote facility. The authors also advise that manuscripts should describe all relevant experimental methods, including sources of experimental animals, experimental procedures used within the animal facility, husbandry details, and any microbiome analysis performed. It is hoped that implementation of these recommendations will facilitate discovery and translation and maximize reward for money and time invested in research.
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 27, ISSUE 3, SEPTEMBER 2016
Article Watch: September 2016 Clive A. Slaughter This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, AU-UGA Medical Partnership, 1425 Prince Ave., Athens, GA 30606, USA. Tel: (706) 713-2216; Fax: (706) 713-2221; E-mail: [email protected], or to any member of the editorial board. Article summaries reflect the reviewer’s opinions and not necessarily those of the association.
METABOLOMICS
Cahill J F, Kertesz V, Weiskittel T M, Vavrek M, Freddo C, Van Berkel G J. Online, absolute quantitation of propranolol from spatially distinct 20- and 40-mm dissections of brain, liver, and kidney thin tissue sections by laser microdissection–liquid vortex capture–mass spectrometry. Analytical Chemistry 88;2016: 6026–6034. This paper describes methodology for mass spectrometric absolute quantification of a drug molecule (propranolol) and its metabolites in tissue sections that provides high spatial resolution. A 10 mm-thick tissue section collected onto a polyethylene naphthalate membrane glass slide is mounted in a commercial laser capture microdissection system. The laser cuts from the tissue-section, squareshaped samples, measuring 20 3 20 mm or 40 3 40 mm. An additional laser pulse aimed at the cut sample causes it to fall into a stream of flowing solvent that transports it to the electrospray (ESI) probe of an online mass spectrometer. The drug is extracted from the tissue dissolving in the solvent stream. The ratio of propranolol signal strength to the signal strength of a fixed concentration of propranolol-d7 spiked into the solvent is measured and compared with a standard curve constructed with known quantities of propranolol. Spatial resolution in this study was limited only by the sensitivity of detection of the analyte: sample sizes as small as 5–10 mm diameter are reported to be achievable with the dissection system. The measurements compare well with values obtained using a tissue punch and HPLC/ESI/mass spectrometry (MS)/MS. The methodology requires no addition of matrix, as would be needed for matrix-assisted laser desorption/ionization (MALDI) MS. The system can, in principle, be used for measuring multiple analytes in a lipidomics-type approach.
doi: 10.7171/jbt.16-2703-005
Journal of Biomolecular Techniques 27:119–124 © 2016 ABRF
CARBOHYDRATES AND GLYCOCONJUGATES
Song X, Ju H, Lasanajak Y, Kudelka M R, Smith D F, Cummings R D. Oxidative release of natural glycans for functional glycomics. Nature Methods 13;2016:528–534. Glycans are generally released from glycoconjugates for the purposes of structural and functional analysis by treatment with expensive enzymes or corrosive or toxic chemicals. Most such procedures are suitable for only small amounts of starting material and are not readily amenable to scaling up. Song et al. note that the use of commercial bleach for sterilization exploits the strong oxidative power of its active ingredient, sodium hypochlorite (NaClO), toward proteins in microorganisms. In the present paper, they therefore explore the use of NaClO for oxidative release of glycans from glycoconjugates as a potentially simple, inexpensive procedure suitable for use on a preparative scale. They show that treatment of various glycoproteins with 1% NaClO degrades the protein moiety within minutes. Free N-glycans are released as glycosamines, which spontaneously convert to free reducing glycans in aqueous solution at room temperature. Otherwise-labile sialic acid residues are well preserved. The glycans can react with amines, allowing addition of fluorescent tags that facilitate chromatographic analysis and purification. The authors demonstrate release of N-glycans from sources that include human saliva and chicken egg whites and yolks and characterize these glycans by MS and 1H NMR. They also show that the fractionated N-glycans immobilized on microarrays are able to bind specific plant lectins. The authors go on to demonstrate that NaClO releases O-glycan from mucins and fetuin, although the greater stability of O-glycosidic linkages requires a higher concentration of NaClO. The glycans released are found to be linked at the reducing end to glycolic acid (presumably derived from serine) or lactic acid (presumably derived from threonine). Finally, the authors show that NaClO releases glycans from glycosphingolipids as glycan nitriles. The nitrile can also be used for fluorescent tagging. Oxidative
SLAUGHTER / ARTICLE WATCH
release of glycans with NaClO is anticipated to facilitate the archiving and characterization of diverse glycans from animal and human sources.
PROTEOMICS
Abelin J G, Patel J, Lu X, Feeney C M, Fagbami L, Creech A L, Hu R, Lam D, Davison D, Pino L, Qiao J W, Kuhn E, Officer A, Li J, Abbatiello S, Subramanian A, Sidman R, Snyder E, Carr S A, Jaffe J D. Reduced-representation phosphosignatures measured by quantitative targeted MS capture cellular states and enable large-scale comparison of drug-induced phenotypes. Molecular & Cellular Proteomics 15;2016:1622–1641. Prominent among cellular responses to perturbation by drugs, stressors, hormones, interactions with extracellular matrix, and contact with neighboring cells are responses mediated by changes in signaling pathways comprised of protein kinases. These pathways affect the phosphorylation state of very large numbers of phosphate acceptor sites on proteins. For example, .70,000 sites of phosphorylation on serine and threonine residues are known in the human proteome. Such a number is far too large to monitor in each of the thousands of samples that one would like to analyze in studies of cell physiology, but only a few hundred protein kinases determine the occupancy of all of these phosphorylation states. This suggests the possibility that occupancy of many of the sites might be coordinately regulated and that the monitoring of a small subset of them might provide an adequate description of the various responses to perturbation that cells can mount. Abelin et al. here investigate this possibility. They study the phosphorylation state of sites in 3 cell lines treated with 26 different bioactive small molecules, cluster the observed responses, and pick representative phosphopeptides from each cluster for monitoring. They develop a targeted parallel reaction monitoring assay to measure 96 of these phosphopeptides. Sample processing is fully automated, so sample throughput is maximized and technical variance minimized. The procedure allowed 95% of the phosphopeptides to be detected in ;200 samples. The results reproduce cell-state classifications previously formulated on the basis of much more extensive datasets acquired in discovery experiments. The performance of the assay is then verified by measuring the same phosphopeptides in new samples generated under new treatment conditions, cell types, and time points. The assay is shown to be sensitive to perturbations in a diverse set of common signaling pathways. This reduced representation of phosphoprofiling assay is believed to be suitable for large-scale 120
phosphoproteome studies involving longitudinal comparisons among thousands of samples and, therefore, is hoped to contribute to an understanding of cellular responses to diverse stimuli. Wilson R E, Groskreutz S R, Weber S G. Improving the sensitivity, resolution, and peak capacity of gradient elution in capillary liquid chromatography with large-volume injections by using temperatureassisted on-column solute focusing. Analytical Chemistry 88;2016:5112–5121. Capillary columns are in widespread use for separating peptides by HPLC in online mass spectrometric analysis. For convenience of sample preparation, sample volumes are often large compared with the volume of the capillary separation bed. This may lead to broadening of peaks for analytes that elute early in a gradient separation. Short, hydrophilic peptides exhibit such broadening during reverse-phase separation. The authors of the present paper offer a remedy for this problem. Because analytes are more strongly retained at lower column temperature, the column is maintained at subambient temperature (27.5°C) during sample loading. The column temperature is then increased quickly to one suitable for chromatographic separation (65°C) before starting gradient elution. The process is fully automated and requires special hardware. It provides significant improvement in detection sensitivity, peak resolution, and peak capacity for small peptides. Shao S, Guo T, Gross V, Lazarev A, Koh C C, Gillessen S, Joerger M, Jochum W, Aebersold R. Reproducible tissue homogenization and protein extraction for quantitative proteomics using MicroPestle-assisted pressure-cycling technology. Journal of Proteome Research 15;2016:1821–1829. Reproducible and efficient extraction of proteins from the small amounts of tissue in clinical biopsy samples is challenging. Automated pressure cycling technology has been developed to meet this need. Samples are subjected to alternating high and low pressure in a Barocycler device for disruption without mechanical shearing. Shao et al. here describe improvements in this technology. A disposable, close-fitting Teflon pestle is inserted into a microtube containing the sample and buffer. During pressure pulses, the tube walls collapse around the incompressible pestle, producing an homogenizer-like effect. The methodology is demonstrated in proteomic studies of mouse liver, heart, brain, and human kidney. JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 27, ISSUE 3, SEPTEMBER 2016
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FUNCTIONAL GENOMICS AND PROTEOMICS
Sadhu M J, Bloom J S, Day L, Kruglyak L. CRISPRdirected mitotic recombination enables genetic mapping without crosses. Science 352;2016: 1113–1116. Linkage and association studies are conducted by associating a phenotype with genetic markers that define a chromosome region. The identification of which specific gene within such a region of interest is responsible for a given phenotype, however, is much more difficult, as it relies on rare recombination events within the region to distinguish the effects of the included genes. Sadhu et al. here use clustered, regularly interspaced, short palindromic repeats (CRISPR) to generate recombination events targeted to predetermined locations within genomic regions of interest for the purposes of fine (high-resolution) genetic mapping. They use the CRISPR-CRISPR associated protein 9 (Cas9) system to induce double-stranded breaks at chosen sites in a chromosome of a diploid cell undergoing mitosis. The homologous chromosome is left intact to serve as a template for repair by homologous recombination, a process that results in loss of heterozygosity (gene conversion). The authors show that CRISPRinduced recombination occurs with high efficiency and with few off-target effects. The authors test their system by generating a recombination panel in yeast cells to map a phenotype (manganese sensitivity) to a polymorphism in a particular transporter (Pmr1). Such trait mapping with targeted loss-of-heterozygosity panels is anticipated to become a standard method for localizing the genetic basis of phenotypic variation. Evers B, Jastrzebski K, Heijmans J P M, Grernrum W, Beijersbergen R L, Bernards R. CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes. Nature Biotechnology 34;2016:631–633. Morgens D W, Deans R M, Li A, Bassik M C. Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes. Nature Biotechnology 34; 2016:634–636. Two groups compare the effectiveness of RNA interference (RNAi) and CRISPR methodology in screening for essential genes in mammalian cultured cells. A similar approach is used by both groups. They use pooled libraries of short hairpin RNA (shRNA) or CRISPR single-guide RNA to look for dropout of gold-standard essential and nonessential genes by high-throughput sequencing. In this JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 27, ISSUE 3, SEPTEMBER 2016
way, they seek to determine the sensitivity and specificity of each methodology. Evers et al. find that CRISPR performs better than RNAi, whereas Morgens et al. find that the 2 methods perform similarly. The 2 groups do conduct testing with RNA libraries of different complexity: for example, the shRNA library of Evers et al. has 4.8 RNA reagents per gene, whereas that of Morgens et al. has 25. Such a difference could explain the superior results observed by Morgens et al. with RNAi. Such differences notwithstanding, the data suggest that the choice of genes for targeting and the choice of RNA screening libraries might affect as big of an influence on outcome as the methodological approach chosen. Of special interest is that both groups observed less than the expected overlap in the list of essential genes detected by RNAi and CRISPR. The mode of action of the 2 methods appears to select for identification of somewhat different sets of genes. The basis for these differences remains to be determined and will be of particular interest to investigators using these methods for loss-of-function and other kinds of assays. Morgens et al. additionally provide computational methods for combining the results of RNAi and CRISPR screens. IMAGING
Morisaki T, Lyon K, DeLuca K F, DeLuca J G, English B P, Zhang Z, Lavis L D, Grimm J B, Viswanathan S, Looger L L, Lionnet T, Stasevich T J. Real-time quantification of single RNA translation dynamics in living cells. Science 352;2016: 1425–1429. Wang C, Han B, Zhou R, Zhuang X. Real-time imaging of translation on single mRNA transcripts in live cells. Cell 165;2016:990–1001. Wu B, Eliscovich C, Yoon Y J, Singer R H. Translation dynamics of single mRNAs in live cells and neurons. Science 352;2016:1430–1435. Yan X, Hoek Tim A, Vale Ronald D, Tanenbaum Marvin E. Dynamics of translation of single mRNA molecules in vivo. Cell 165;2016:976–989. Four groups have explored methods for imaging nascent polypeptides during synthesis on single mRNA molecules within living cells. To visualize polypeptides as they emerge from the ribosome, all 4 groups incorporate tandem repeats of a polypeptide epitope that rapidly bind pre-existing fluorescent antibodies following ribosomal assembly and use fluorescent RNA-binding proteins to detect colocalized, single mRNA molecules. The imaging studies enabled by these techniques reveal initiation rates, 121
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elongation speeds, and ribosome spacing on polysomes in a cellular context. By focusing on individual mRNA molecules, they also reveal spatial and temporal variation in translational activity, gene-specific effects, and characteristics of intracellular polysome mobility. The present studies used exogenous message constructs, but similar approaches based on genome editing could be used to reveal details of tissue-specific translational control during development. Ngo J T, Adams S R, Deerinck T J, Boassa D, Rodriguez-Rivera F, Palida S F, Bertozzi C R, Ellisman M H, Tsien R Y. Click-EM for imaging metabolically tagged nonprotein biomolecules. Nature Chemical Biology 12;2016:459–465. Ngo et al. describe a technique for electron microscope (EM) imaging of selected nonprotein biomolecules, such as glycans, nucleic acids and lipids, at nanometer resolution. They adopt a metabolic labeling approach in which metabolic analogs of monosaccharides, nucleotides, or fatty acids containing a bio-orthogonal functional group are provided to cells for incorporation into biomolecules by endogenous biosynthetic processes. The functional group is then targeted for attachment of a fluorescent dye by click chemistry. The dye generates singlet oxygen upon photolysis, and the singlet oxygen mediates local polymerization of diaminobenzidine to form a polymeric precipitate that can be stained with osmium tetroxide for visualization by EM. The dye-labeled biomolecules can also be imaged by fluorescence microscopy for correlation with the EM images. The authors use this procedure to image DNA, RNA, and lipids in cultured cells and neurons and to track peptidoglycan synthesis in a grampositive bacterium. The methodology does not involve permeabilization of cells, which might disrupt ultrastructural features. The methodology also introduces reagents required for EM contrast only after fixing the cells. It enables high resolution by ensuring that the deposition of contrast materials occurs within nanometers of the structures of interest. The authors envision application of the technique to studying structures that are difficult to resolve by light microscopy, such as axon terminals, mitochondria, synaptic vesicles, and autophagosomes. Wu Q, Comi T J, Li B, Rubakhin S S, Sweedler J V. On-tissue derivatization via electrospray deposition for matrix-assisted laser desorption/ionization mass spectrometry imaging of endogenous fatty acids in rat brain tissues. Analytical Chemistry 88;2016:5988–5995. Imaging by MALDI is of limited efficacy for molecules that are small enough to incur interference by matrix ions, 122
molecules of low abundance, and molecules that ionize poorly. Wu et al. here describe a method for MALDI imaging of free fatty acids, a class of biomolecules that includes species of interest for the synthesis of signaling molecules and lipid metabolism but that ionizes poorly in the positive ion mode. They enhance the detectability of these molecules by specific derivatization with 2-picolylamine in the presence of 2,2-dipyridyl disulfide and triphenylphosphine. This adds a pyridyl moiety to the carboxyl group via amide linkage, promoting formation of positive ions. The reaction occurs rapidly over a broad temperature range, making it suitable for derivatization on tissue slices. Importantly, the derivatization reagent is applied by electrospray deposition. Electrospray produces charged, nanoscale droplets that promote extraction and derivatization of target fatty acids but minimizes their delocalization. With the use of this procedure, the authors visualized 6 fatty acids in selected regions of the rat brain with effective spatial resolution of 20 mm and were able to distinguish fatty acid distribution in adjacent cell layers of the hippocampus. They expect that electrospray-assisted derivatization will prove useful for imaging further classes of analyte. CELL BIOLOGY AND TISSUE ENGINEERING
Shahbazi M N, Jedrusik A, Vuoristo S, Recher G, Hupalowska A, Bolton V, Fogarty N M E, Campbell A, Devito L G, Ilic D, Khalaf Y, Niakan K K, Fishel S, Zernicka-Goetz M. Self-organization of the human embryo in the absence of maternal tissues. Nature Cell Biology 18;2016:700–708. Deglincerti A, Croft G F, Pietila L N, ZernickaGoetz M, Siggia E D, Brivanlou A H. Selforganization of the in vitro attached human embryo. Nature 533;2016:251–254. During human embryonic development, the fertilized egg divides to form a free-floating blastocyst consisting of 3 cell lineages: the epiblast, hypoblast, and trophectoderm. Following blastocyst implantation into the uterine wall at d 5, these lineages produce the fetus, extraembryonic yolk sac, and placenta, respectively. Study of the remodeling that occurs in human embryos at implantation has hitherto been impeded by lack of suitable in vitro culture conditions. (The value of experiments with nonhuman animals is limited by significant differences between species in timing and control of the events.) Two groups now describe culture conditions that permit in vitro observation throughout the implantation stages. They show that blastocysts, derived from supernumerary fertilized eggs, attach to the culture dish. The attached blastocysts exhibit key transitions, including JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 27, ISSUE 3, SEPTEMBER 2016
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segregation of embryonic and extraembryonic cell lineages, epiblast polarization, formation of the proamniotic cavity and bilaminar disk, appearance of a presumptive yolk sac, and differentiation of the trophoblast into the cytotrophoblast and syncytiotrophoblast. The occurrence of these transitions in the absence of maternal tissues highlights the self-organizing capability of the human embryo. The improved technical capabilities described in these papers are expected to help understand the signaling processes that mediate normal implantation and its accompanying morphogenetic events and to gain insight into implantation failure, which is a major cause of early pregnancy loss. Both groups terminated their experiments at d 14 postfertilization (before formation of the primitive streak and the embryo germ layers) in compliance with long-standing international policy adopted to permit embryo research within ethical guidelines. It is likely that the present technical advances will prompt re-evaluation of this policy. (For analysis of policy and ethical considerations, see Hyun I, Wilkerson A, Johnson J. Nature 533;2016:170–171.) Hyslop L A, Blakeley P, Craven L, Richardson J, Fogarty N M E, Fragouli E, Lamb M, Wamaitha S E, Prathalingam N, Zhang Q, O’Keefe H, Takeda Y, Arizzi L, Alfarawati S, Tuppen H A, Irving L, Kalleas D, Choudhary M, Wells D, Murdoch A P, Turnbull D M, Niakan K K, Herbert M. Toward clinical application of pronuclear transfer to prevent mitochondrial DNA disease. Nature 534;2016:383–386. Hyslop et al. report preclinical studies of reproductive techniques to reduce the risk of human disease resulting from mutation in mitochondrial DNA. The method involves transplanting nuclei from oocytes with affected mitochondria to enucleated donor oocytes free of abnormalities in mitochondrial DNA. Initial studies indicated that nuclear transplantation between human oocytes that are arrested in meiosis division II results in a high incidence of abnormal fertilization. An alternative approach proves to be more successful. It involves transplanting the maternal and paternal genomes, separately packaged in discrete pronuclei, after in vitro fertilization. The authors find that this transplantation procedure gives best results when performed shortly after the completion of the second meiotic division, rather than shortly before the first mitotic division that produces a 2-cell embryo. This method results in improved survival and produces blastocysts of good morphologic quality. No effect on aneuploidy or gene expression is detected, and carryover of mitochondrial DNA is low. These preliminary results encourage further exploration of the technology JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 27, ISSUE 3, SEPTEMBER 2016
for reducing the risk of mitochondrial disease in the children of affected mothers. Treutlein B, Lee Q Y, Camp J G, Mall M, Koh W, Shariati S A M, Sim S, Neff N F, Skotheim J M, Wernig M, Quake S R. Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq. Nature 534;2016:391–395. Mouse embryonic fibroblasts can be directly reprogrammed to form induced neuronal cells by overexpression of 3 transcription factors: Ascl1, Brn2, and Myt1l. Transdifferentiation occurs within 2–3 wk in culture at an efficiency of up to 20%. Ascl1 alone also mediates transdifferentiation, but with lower efficiency. The present paper shows how single-cell RNA sequencing (RNA-Seq) can be used to analyze the molecular pathways by which transdifferentiation occurs and to identify processes that limit its efficiency. To reconstruct the progress of cells toward the neuronal phenotype under Ascl1 overexpression, the authors characterize the transcriptome of a total of 405 individual cells representing 0, 2, 5, and 22 d in culture. They find that the initial response, in terms of Ascl1 expression and silencing of fibroblast-associated genes, is relatively homogeneous among fibroblasts, with no evidence for variation in susceptibility to transdifferentiation. However, later, they observe cells that show Ascl1 silencing and emergence of a competing differentiation program that activates muscle-like (myogenic) genes, even though these cells turn on the neuronal marker, Tau. Overexpression of Brn2 and Myt1l, in addition to Ascl1, apparently suppresses the competing pathway. This study provides methodology by which the molecular processes responsible for reprogramming and differentiation stability may be studied. It also indicates the importance of using more than 1 marker (such as Tau) for identifying a differentiated state and the potential importance of using multiple transcription factors for directing reprogramming. MICROBIOME
Browne H P, Forster S C, Anonye B O, Kumar N, Neville B A, Stares M D, Goulding D, Lawley T D. Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation. Nature 533;2016:543–546. The bacterial assemblage that inhabits our gastrointestinal (GI) tract comprises complex ecological communities known as microbiota. Microbiota typically contain 100–1000 bacterial species. The recent increase in our knowledge of microbiota species diversity has relied heavily 123
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on methodology involving metagenomic sequencing that does not require the bacteria to be cultured. Indeed, it is generally believed that the majority of our commensal GI bacteria is recalcitrant to culturing. Browne et al. here demonstrate how a substantial majority of these bacteria is, in fact, amenable to culturing. The authors homogenize fecal samples from healthy human donors, serially dilute the homogenate, and inoculate it onto agar plates containing a complex, broad-range bacteriologic medium. Single colonies are then streaked to purity. All of these manipulations are conducted under anaerobic conditions, as GI bacteria are typically very sensitive to ambient oxygen. The pure colonies may then be characterized by genomic sequencing. Comparison of the sequences with the results of metagenomic sequencing of the original unfractionated feces indicates that 74% of the computationally derived metagenomic species in the unfractionated samples are represented in the pure cultures. The authors proceed to isolate, sequence, and archive 137 bacterial species in this way, including 45 presumptively novel species, 20 novel genera, and 2 novel families. Because spores of the pathogen Clostridium difficile are resistant to ethanol—a commonly used disinfectant—the authors use their system to characterize spore-forming bacteria identified as such by their ethanol resistance. The results indicate that 50–60% of the bacterial genera in healthy individuals produce spores. By sequencing, the authors define 66 conserved genes linked to the ethanol-resistant phenotype that may be used to predict sporulation capability in new bacterial isolates. The finding also helps to explain transmission of bacteria from 1 person to another. The capability to culture and sequence the genomes of substantial numbers of bacterial species from the human GI tract is expected to support important advances in knowledge of our commensal partners.
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Stappenbeck T S, Virgin H W. Accounting for reciprocal host–microbiome interactions in experimental science. Nature 534;2016:191–199. It is increasingly apparent that variation in microbiota can affect the incidence and course of diseases, the physiologic processes on which diseases depend, and the results of treatment. Whereas the effects of GI microbiota on the intestinal epithelium are generally appreciated, awareness of the effects on other organ systems of commensals, located either in the GI tract or elsewhere, is weaker. For example, apparently heritable characteristics might be mediated by vertical transmission of microbes, as well as by transmission of host genes, and effects of age, circadian changes, and exposure to pathogens might all be strongly affected by commensal microbes. The effects of variables related to microbiota may lead to erroneous interpretation of experimental results if experimental design fails to control for them and may create confusion as a result of unreproducible findings. The authors of the present perspective suggest features of experimental design to mitigate such risks. For example, investigators using mouse models are advised to compare phenotypes of test and control animal groups that have been raised as littermates rather than to use controls purchased from a remote facility. The authors also advise that manuscripts should describe all relevant experimental methods, including sources of experimental animals, experimental procedures used within the animal facility, husbandry details, and any microbiome analysis performed. It is hoped that implementation of these recommendations will facilitate discovery and translation and maximize reward for money and time invested in research.
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