ARTICLE WATCH
Article Watch: July 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, MCG-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.
DNA SEQUENCING AND GENOTYPING
Quick J, Loman N J, Duraffour S, Simpson J T, Severi E, Cowley L, Bore J A, Koundouno R, Dudas G, Mikhail A, Ou´edraogo N, Afrough B, Bah A, Baum J H J, Becker-Ziaja B, Boettcher J P, Cabeza´ Carter L L, Cabrerizo M, Camino-S´a nchez A, Doerrbecker J, Enkirch T, Dorival I G, Hetzelt N, Hinzmann J, Holm T, Kafetzopoulou L E, Koropogui M, Kosgey A, Kuisma E, Logue C H, Mazzarelli A, Meisel S, Mertens M, Michel J, Ngabo D, Nitzsche K, Pallasch E, Patrono L V, Portmann J, Repits J G, Rickett N Y, Sachse A, Singethan K, Vitoriano I, Yemanaberhan R L, Zekeng E G, Racine T, Bello A, Sall A A, Faye O, Faye O, Magassouba N F, Williams C V, Amburgey V, Winona L, Davis E, Gerlach J, Washington F, Monteil V, Jourdain M, Bererd M, Camara A, Somlare H, Camara A, Gerard M, Bado G, Baillet B, Delaune D, Nebie K Y, Diarra A, Savane Y, Pallawo R B, Gutierrez G J, Milhano N, Roger I, Williams C J, Yattara F, Lewandowski K, Taylor J, Rachwal P, J. Turner D, Pollakis G, Hiscox J A, Matthews D A, Shea M K O, Johnston A M, Wilson D, Hutley E, Smit E, Di Caro A, W¨olfel R, Stoecker K, Fleischmann E, Gabriel M, Weller S A, Koivogui L, Diallo B, Ke¨ıta S, Rambaut A, Formenty P, G¨unther S, Carroll M W. Real-time, portable genome sequencing for Ebola surveillance. Nature 530;2016: 228–232. The recent Ebola epidemic in West Africa presented an opportunity for genome sequencing to contribute to epidemiologic tracking of the infectious agent and monitoring responses to vaccines and treatments. This paper documents the deployment of field stations in local, resource-poor locations to perform genomic surveillance of the Ebola virus. DNA sequencing was performed with the MinION
doi: 10.7171/jbt.16-2702-005
instrument from Oxford Nanopore Technologies (Oxford, United Kingdom), a hand-held device weighing ,100 g that monitors sequence-dependent fluctuations in electrical current as a DNA strand passes through a protein nanopore. The principle of operation enables long sequence reads, although its low accuracy requires consensus averaging of many reads to achieve adequate sequence accuracy. The authors transported to West Africa 3 MinION sequencers, 4 laptop computers, a thermocycler, heat-block, pipettes, and sufficient reagents and consumables to perform sequencing. The entire package weighed ,50 kg. They started sequencing within 2 d of arrival in Guinea. The genome sequencing workflow could be accomplished in ,24 h, although the protocol was usually performed over 2 d. Sufficient reads were often accomplished in 1 h of sequencing time. The authors sequenced 142 samples over 148 MinION runs during a 6 mo period and were able to document the lineage history of Ebola virus in the area. Their main logistic obstacles related to lack of continuity in main electrical supplies, which affected the thermocyclers but not the MinION, and difficulties with internet connectivity, which was needed for data analysis. The results of this study validate the practicality of genome sequencing in resource-limited settings for monitoring outbreaks of infectious disease. Lehmann-Werman R, Neiman D, Zemmour H, Moss J, Magenheim J, Vaknin-Dembinsky A, Rubertsson S, Nellg˚a rd B, Blennow K, Zetterberg H, Spalding K, Haller M J, Wasserfall C H, Schatz D A, Greenbaum C J, Dorrell C, Grompe M, Zick A, Hubert A, Maoz M, Fendrich V, Bartsch D K, Golan T, Ben Sasson S A, Zamir G, Razin A, Cedar H, Shapiro A M J, Glaser B, Shemer R, Dor Y. Identification of tissue-specific cell death using methylation patterns of circulating DNA. Proceedings of the National Academy of Sciences USA 113;2016: E1826–E1834. Lehmann-Werman et al. here demonstrate proof of principal that cell-free, circulating DNA released from dying Journal of Biomolecular Techniques 27:84–89 © 2016 ABRF
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cells may be used to identify the tissue of DNA origin. Their methodology relies on detection of cell type-specific methylation markers. Serum or plasma from donors is treated with bisulfite, PCR amplified, and sequenced to quantify epigenetic markers of interest. The authors identify pancreatic b-cell DNA in patients with recently diagnosed type 1 diabetes and in recipients of islet grafts; oligodendrocyte DNA in patients with relapsing multiple sclerosis; neuronal or glial DNA in patients who have recently suffered traumatic brain injury or cardiac arrest; and exocrine pancreatic DNA in patients with pancreatitis or pancreatic cancer. As levels of cell-free DNA from affected tissues are low in the early stages of disease, the methodology is unlikely to contribute to diagnosis for reasons related to detection sensitivity but is hoped to contribute to an understanding of normal tissue dynamics and to provide an alternative approach to monitoring disease progression and treatment efficacy. MOLECULAR SYNTHESIS AND SYNTHETIC BIOLOGY
Hutchison C A, Chuang R-Y, Noskov V N, AssadGarcia N, Deerinck T J, Ellisman M H, Gill J, Kannan K, Karas B J, Ma L, Pelletier J F, Qi Z-Q, Richter R A, Strychalski E A, Sun L, Suzuki Y, Tsvetanova B, Wise K S, Smith H O, Glass J I, Merryman C, Gibson D G, Venter J C. Design and synthesis of a minimal bacterial genome. Science 351;2016:aad6253. This paper is a report on progress at the J. Craig Venter Institute (JCVI) toward defining the minimal, fully synthetic genome needed to specify an autonomously replicating cell. Previously, the group accomplished total chemical synthesis of the 1079 kilobase pair (kbp) genome of Mycoplasma mycoides, which encodes 901 proteins, and introduced it into cells of a different mycoplasma species, Mycoplasma capricolum, under conditions in which the genome of M. capricolum is lost, and only the transplanted genome remains. The resulting strain, JCVI-syn1.0, is subjected to genome minimization in this paper. The result is a strain called JCVI-syn3.0 that has a genome of just 531 kbp encoding 473 proteins and 35 annotated RNAs. Initial attempts to design the minimal genome de novo based on available knowledge of molecular biology failed to produce viable cells, suggesting that current knowledge is incomplete. An alternative approach was to divide up the JCVIsyn1.0 genome into 8 independent modules, delete pieces of each or disrupt function by transposon insertion, and then reassemble the genome for testing cell viability. This approach succeeded in producing a strain with doubling time as rapid as 3 h—a finding of interest, as there is a tradeJOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 27, ISSUE 2, JULY 2016
off between genome size and growth rate in small genomes subject to minimization. Most of the genes that remain included are involved in gene expression, membrane biogenesis, and metabolism and to a lesser extent, genome replication and preservation of genomic information. Most intriguingly, 79 genes (17%) remain unassigned to a category, and 149 are of unknown function. Investigation of these genes may reveal new cellular processes. The authors’ line of synthetic biology research is of fundamental interest in cell and molecular biology. Spin-off methods may prove useful in designing genomes for industrial biosynthesis and therapeutic intervention. MASS SPECTROMETRY
Riley N M, Mullen C, Weisbrod C R, Sharma S, Senko M W, Zabrouskov V, Westphall M S, Syka J E P, Coon J J. Enhanced dissociation of intact proteins with high capacity electron transfer dissociation. Journal of The American Society for Mass Spectrometry 27;2015:520–531. Electron transfer dissociation (ETD) is a method with favorable characteristics for acquiring amino acid sequence information from intact proteins, as it works well with multiply charged precursor ions and minimizes cleavage of labile side chains and post-translational modifications. The implementation of intact protein ETD in ion-trapping instruments, however, is impeded by the limited charge capacity of ion traps, because as the size of the protein precursor increases, its charge state also increases, and the number of precursor ions that can be stored diminishes proportionately. At the same time, product ion spectra become more complex, and signal is split between increasing numbers of fragments. Riley et al. here describe modifications to the newest generation of quadrupole Orbitrap linear ion trap mass spectrometers, the Orbitrap Fusion Lumos (Thermo Fisher Scientific, San Jose, CA, USA). The modifications increase the instrument’s precursor-trapping capacity for use in analysis of intact proteins. As in previous versions of the instrument, precursors are sequestered in the dual-cell quadrupole linear ion trap mass-to-charge ratio analyzer (A-QLT) before reaction with ETD reagent anions, but the location within the A-QLT, where the ions are stored, is altered here to permit storage of larger numbers of precursor cations. This enables enhancement of product ion signal-to-noise ratios for a given acquisition time, with attendant higher-sequence coverage and less need for signal averaging. The modifications are now available on a commercial platform for analysis of intact proteins and top-down proteomics. 85
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Fukuyama Y, Nakajima C, Izumi S, Tanaka K. Membrane protein analyses using alkylated trihydroxyacetophenone (ATHAP) as a MALDI matrix. Analytical Chemistry 88;2016:1688–1695. This work is an extension of previous studies describing a new matrix for matrix-assisted laser desorption/ ionization (MALDI)-mass spectrometry analysis of hydrophobic peptides from membrane proteins: (2,4,6trihydroxyphenyl)octan-1-one (ATHAP), an alkylated version of trihydroxyacetophenone that incorporates an alkyl chain. which confers increased affinity for hydrophobic peptides. The present report extends the use of ATHAP to detection of intact molecular ions of bacteriorhodopsin, a protein with 7 transmembrane domains that is difficult to detect using a-cyano-4-hydroxycinnamic acid or sinapinic acid matrices. ATHAP also supports detection of peptides containing hydrophobic sequences from a variety of other membrane proteins, thereby confirming its general suitability for their analysis. McMillen C L, Wright P M, Cassady C J. Negative ion in-source decay matrix-assisted laser desorption/ ionization mass spectrometry for sequencing acidic peptides. Journal of The American Society for Mass Spectrometry 27;2016:847–855. McMillen et al. study conditions for MALDI in-source decay to acquire amino acid sequence information for deprotonated acidic peptides in negative ion mode. They test 8 acidic peptides ranging from 11 to 33 residues with each of 7 different matrices and identify 1,5-diaminonaphthalene as the matrix supporting the best fragmentation. As in ETD, electron capture dissociation, and MALDI in-source decay in the positive ion mode, product ions resulting from cleavage of the N-Ca bonds predominate. Negative ion in-source decay is expected to be a generally useful method for analysis of acidic peptides. METABOLOMICS
Hensley Christopher T, Faubert B, Yuan Q, LevCohain N, Jin E, Kim J, Jiang L, Ko B, Skelton R, Loudat L, Wodzak M, Klimko C, McMillan E, Butt Y, Ni M, Oliver D, Torrealba J, Malloy Craig R, Kernstine K, Lenkinski Robert E, DeBerardinis Ralph J. Metabolic heterogeneity in human lung tumors. Cell 164;2016:681–694. The authors here combine metabolic labeling with [U-13C]glucose and clinical imaging by dynamic contrastenhanced magnetic resonance imagery (DCE-MRI) and 86
fluoro-2-deoxyglucose positron emission tomography (FDG-PET) to assess metabolic heterogeneity within and between non-small cell lung cancers in human patients undergoing tumor resection. Metabolic labeling provides information about the metabolic fuel(s) preferred by the malignant tissue, DCE-MRI measures tumor perfusion, and FDG-PET shows cellular uptake of glucose. Compared with normal lung tissue, the tumors display both enhanced glycolysis and enhanced tricarboxylic acid cycle (TCA) activity. This is different from the switch from oxidative to glycolytic metabolism characteristic of the Warburg effect. The enhanced glucose uptake shown by FDGPET apparently supports up-regulated flux through both pathways. The authors observe that entry of 13C into the TCA cycle is dominated by pyruvate dehydrogenase. They also detect low enrichment of 13C in acetyl CoA, suggesting that the tumor cells are oxidizing nutrients other than glucose—probably lactate. Metabolic variability in 13C enrichment is observed and is closely correlated with local perfusion. Limited perfusion is linked to high 13C enrichment, suggesting that cells increase their reliance on glucose compared with other substrates. These results illustrate how imaging combined with metabolic labeling can be used to characterize metabolic heterogeneity in tumors. 18
PROTEINS—PURIFICATION AND CHARACTERIZATION
Gault J, Donlan J A C, Liko I, Hopper J T S, Gupta K, Housden N G, Struwe W B, Marty M T, Mize T, Bechara C, Zhu Y, Wu B, Kleanthous C, Belov M, Damoc E, Makarov A, Robinson C V. Highresolution mass spectrometry of small molecules bound to membrane proteins. Nature Methods 13; 2016:333–336. Gault et al. here describe methods for mass spectrometric analysis of intact membrane proteins in which a modified Orbitrap mass spectrometer provides sufficient resolution to discriminate the binding of lipids, detergents, peptides, and drugs. Protein complexes are protected by micelles during translation into the gas phase. Micelles are removed by collisional activation in the higher-energy collisional dissociation cell or in the source region or both. Voltage gradients are chosen to control the release of the protein without disrupting its stoichiometry or interaction with ligands. The authors characterize a variety of membrane proteins, ranging from 26 to 186 kDa. They measure protein–ligand association constants, deconvolute concomitant binding of drug and lipid molecules, and resolve bound lipids from the same class but having different chain length. The direct identification of bound JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 27, ISSUE 2, JULY 2016
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lipids in this way represents a significant advance over existing mass spectrometric methods. Ayyer K, Yefanov O M, Oberth¨ur D, RoyChowdhury S, Galli L, Mariani V, Basu S, Coe J, Conrad C E, Fromme R, Schaffer A, D¨orner K, James D, Kupitz C, Metz M, Nelson G, Xavier P L, Beyerlein K R, Schmidt M, Sarrou I, Spence J C H, Weierstall U, White T A, Yang J-H, Zhao Y, Liang M, Aquila A, Hunter M S, Robinson J S, Koglin J E, Boutet S, Fromme P, Barty A, Chapman H N. Macromolecular diffractive imaging using imperfect crystals. Nature 530;2016:202–206. The sharp, regularly spaced intensity peaks that arise when X-rays are diffracted by the molecules in a wellordered crystal lattice result from constructive interference between the diffracted rays. The resolution at which molecular structure can be reconstructed depends on the Bragg diffraction limit, which is determined, in part, by the regularity of molecular spacing in the crystal lattice. Disorder decreases the intensity of the sharp Bragg intensity peaks and produces continuous modulation of these diffraction intensities. This gives the appearance of light and shadowy regions in the diffraction pattern, an effect seen all too often from imperfect protein crystals. Ayyer et al. have found a way to use this continuous modulation to enhance the structural information obtained by X-ray diffraction. By treating the continuous diffraction pattern as the incoherent sum of diffraction from rigid individual molecules aligned along several discrete crystallographic orientations, they derive more information than the sharp Bragg intensities provide alone. They first use the Bragg intensities to calculate an electron density map at low resolution. Then, they use the continuous diffraction pattern to refine the map to a resolution beyond the Bragg diffraction limit. In this way, they achieve an improvement in the resolution attained for the photosystem II dimer, a very large membrane protein of molecular weight 700 kDa, ˚ It is hoped that the approach will be from 4.5 to 3.5 A. generally applicable for enhancing resolution from macromolecules that form crystals of imperfect quality. PROTEOMICS
Hart-Smith G, Yagoub D, Tay A P, Pickford R, Wilkins M R. Large scale mass spectrometry-based identifications of enzyme-mediated protein methylation are subject to high false discovery rates. Molecular and Cellular Proteomics 15;2016:989–1006. Special functional significance has been attributed to the post-translational methylation of histones. However, JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 27, ISSUE 2, JULY 2016
methylation occurs widely among proteins, and the location of methylation sites (most commonly on lysine and arginine residues) is of more general interest. Hart-Smith et al. here illustrate how to perform rigorous validation of methylation sites through heavy methyl stable isotope labeling in cell culture. In studies of yeast cells, they provide 13CD3-labeled methionine in the culture medium. Methionine is the precursor of S-adenosyl-L-methionine, the methyl donor used by all known methyltransferases. The resulting mass shifts of methylated peptides allow the number of incorporated methyl groups to be counted. Sources of artifactual methylation include the esterification of glutamic and aspartic acids during sample preparation protocols that use methanol or isopropanol. These reactions may be mistaken for enzyme-mediated monoor dimethylation of proximal arginine or lysine residues during automated spectrum-matching searches, but are distinguished from cellular methylation by the labeling procedure. The authors show that false discovery rates estimated by targetdecoy approaches greatly underestimate actual rates of false discovery of methylation sites. This effect is attributed to the large number of amino acid combinations capable of producing peptide sequences that are isobaric with methylated peptides of different sequence. The rate of false discovery remains high even when antibody-based purification of methylated peptides is used. Therefore, the authors caution against reliance upon target-decoy estimates in studies of protein methylation. Moore S M, Hess S M, Jorgenson J W. Extraction, Enrichment, solubilization, and digestion techniques for membrane proteomics. Journal of Proteome Research 15;2016:1243–1252. This paper revisits the optimal conditions for preparing membrane proteins for proteomic analysis. In studying yeast cells, the authors see optimal numbers of identified proteins, best quantitative recovery of most proteins, and greatest convenience in a protocol that incorporates cell disruption in a French press, no further enrichment step, solubilization with 1% sodium deoxycholate while applying sonication, and digestion with trypsin at 37°C. Cell lysis by sonication, cryopulverization, freeze/thawing, or bead beating provided no advantage. Enrichment by chloroform/methanol partitioning or acetone precipitation resulted in loss of some proteins. Solubilization with methanol and use of enzymes other than trypsin were also not advantageous. FUNCTIONAL GENOMICS AND PROTEOMICS
Kleinstiver B P, Pattanayak V, Prew M S, Tsai S Q, Nguyen N T, Zheng Z, Joung J K. High-fidelity CRISPR–Cas9 nucleases with no detectable genomewide off-target effects. Nature 529;2016:490–495. 87
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Slaymaker I M, Gao L, Zetsche B, Scott D A, Yan W X, Zhang F. Rationally engineered Cas9 nucleases with improved specificity. Science 351;2016:84–88. Genome editing with the clustered, regularly interspaced, short palindromic repeat (CRISPR)-associated 9 (Cas9) protein, used in conjunction with a single-guide RNA (sgRNA), relies on double-stranded DNA cleavage at a site with base sequence complementary to the sgRNA. Cas9 shows high-sequence specificity of target cleavage, even in mammalian genomes, which are some 18003 larger than the bacterial genomes in which the Cas9 protein originally evolved to excise viral DNA. Nevertheless, nonspecific binding by Cas9 is measureable, and off-target cleavage occurs occasionally. The engineering of Cas9 with improved sequence selectivity is now an important goal, as Cas9-based gene editing for clinical purposes is within view. Two groups here describe improvements in Cas9 specificity. Both groups exploit knowledge that the affinity of binding between Cas9-sgRNA and its target has a sequence-specific component and a sequence-nonspecific component that results from interactions between protein and DNA backbone. Both groups approach specificity enhancement by mutating amino acid residues contributing to the nonspecific component of the total binding affinity. With the use of Cas9 from Streptococcus pyrogenes, Kleinstiver et al. mutate residues that contact the backbone of the DNA target strand, whereas Slaymaker et al. mutate residues that contact the complementary DNA single strand. Although overall binding affinity is weakened in both approaches, the difference between onand off-target affinity is broadened. Both groups show that the overall weakening is not so great as to impair on-target nuclease activity significantly, but they demonstrate improved specificity by unbiased detection of cleavage using the most sensitive available high-throughput sequencing methods. The new proteins are expected to speed genome-editing applications in basic science and hasten clinical application. Rajagopal N, Srinivasan S, Kooshesh K, Guo Y, Edwards M D, Banerjee B, Syed T, Emons B J M, Gifford D K, Sherwood R I. High-throughput mapping of regulatory DNA. Nature Biotechnology 34;2016:167–174. Korkmaz G, Lopes R, Ugalde A P, Nevedomskaya E, Han R, Myacheva K, Zwart W, Elkon R, Agami R. Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9. Nature Biotechnology 34;2016:192–198. The human genome is believed to contain .500,000 enhancers (DNA sites at which transcription factors bind 88
and regulate the transcription of sometimes quite-distantly located genes through chromatin looping). The need to grapple with the phenomenal complexity that this number of enhancers represents is highlighted by ongoing reports of their involvement in diseases, such as cancer and autoimmunity, and the therapeutic potential of interfering with the function of proteins, including transcription factors, with which they interact. Existing high-throughput methods for analyzing enhancers involve cloning regulatory elements into plasmid vectors, but this approach does not capture the function of enhancers in their native genomic context. The present 2 papers initiate the use of the CRISPR-Cas9 system to conduct high-throughput mapping of regulatory elements at single-base resolution in the native context. Rajagopal et al. study the regulatory elements that control expression of 4 embryonic stem cell-specific genes, identify proximal and distal regulatory elements necessary for their expression, and discover regulatory elements unmarked by typical epigenetic or chromatin features. Korkmaz et al. describe 2 genetic screens to identify enhancer elements that bind TP53 (tumor protein 53) and estrogen receptor a, identify several endogenous enhancer elements, and characterize domains essential for their activity. These studies represent a new milestone in highthroughput functional screening of regulatory elements in their native context. Kline M C, Romsos E L, Duewer D L. Evaluating Digital PCR for the quantification of human genomic DNA: accessible amplifiable targets. Analytical Chemistry 88;2016:2132–2139. Digital PCR (dPCR) is a method for measuring the quantity of a particular DNA target sequence in a given sample. The sample is partitioned into small volumes (chambers or equal-sized droplets) so that a given volume contains 0 to a few target sequences. The volumes are subjected to a predetermined number of PCR amplification cycles and then scored for the presence or absence of fluorescence signal from the target. The number of volumes that contain at least 1 target sequence relative to the total number of volumes scored is described by the Poisson distribution and provides a measure of the concentration of target sequences in the original sample. The measurement does not depend on comparison with a DNA quantification standard. Rather, it can be used to create reference standards as calibrants for a variety of applications. Kline et al. here report a source of bias in dPCR measurements. They show that dPCR measures not the absolute number of targets in a sample but rather, the number of targets that are accessible and amenable to amplification. Untreated DNA is often treated with endonuclease to render targets accessible, but nucleases can destroy, as well as reveal, targets. If nucleases JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 27, ISSUE 2, JULY 2016
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are to be used, the fraction of targets they make inaccessible must be measured independently. Nucleases giving mean fragment lengths of several thousand base pairs are shown to do less damage than those that cut more frequently. When estimating the amount of human genomic DNA, several different targets should be measured, and the results obtained with different dPCR platforms (chamber and droplet) should be compared to check for agreement. Accuracy further depends on assessment of chamber or droplet volume, which is presently difficult to achieve. These results and recommendations will be of interest to all investigators using dPCR in their research.
hydrochloride (Benadryl), lidocaine hydrochloride, diazepam (Valium), and fluoxetine hydrochloride (Prozac). All drug products are formulated to meet standards set forth by U.S. Pharmacopeia. The formulations are concentrated aqueous or alcohol-based solutions ready for dilution when needed. Solid formulations, such as tablets, however, would require modules for drying, powder transport, solids blending, and tableting that are still under development. With appropriate U.S. Food and Drug Administration approvals, the technology incorporated into the instrumentation described in this paper is expected to enable local production of medications for hospitals, healthcare organizations, pharmaceutical development, and humanitarian aid.
DRUG DESIGN AND CHARACTERIZATION
Adamo A, Beingessner R L, Behnam M, Chen J, Jamison T F, Jensen K F, Monbaliu J-C M, Myerson A S, Revalor E M, Snead D R, Stelzer T, Weeranoppanant N, Wong S Y, Zhang P. Ondemand continuous-flow production of pharmaceuticals in a compact, reconfigurable system. Science 352;2016:61-67. Drug companies generally make pharmaceuticals in large batches using procedures that require large manufacturing plants, protracted periods of time, and extensive storage facilities. Drug shortages are often the result of limitations inherent in such batch-wise manufacturing. The authors of the present paper have developed a continuous manufacturing platform that combines synthesis and drug product formulation into a single unit the size of a refrigerator. It uses continuous flow to streamline, automate, and integrate synthesis processes at the grams/hour scale. The instrument is comprised of reconfigurable modules: an upstream unit houses feeds, pumps, reactors, separators, and pressure regulators for chemical synthesis of an active pharmaceutical ingredient (API) from simple chemical precursors, and a downstream unit contains precipitation tanks, crystallizers, and filters for purification and formulation of the drug product. Elevated pressures and temperatures are used to accelerate reactions, and reagent concentrations are kept high to minimize waste production. An in-line attenuated total reflection Fourier transform infrared spectrometer provides realtime monitoring of the APIs formed. The modules are reconfigured to accomplish synthesis of 4 products encompassing a range of chemical complexity: diphenhydramine
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 27, ISSUE 2, JULY 2016
CELL BIOLOGY AND TISSUE ENGINEERING
Fuzik J, Zeisel A, Mate Z, Calvigioni D, Yanagawa Y, Szabo G, Linnarsson S, Harkany T. Integration of electrophysiological recordings with single-cell RNA-seq data identifies neuronal subtypes. Nature Biotechnology 34;2016:175–183. Cadwell C R, Palasantza A, Jiang X, Berens P, Deng Q, Yilmaz M, Reimer J, Shen S, Bethge M, Tolias K F, Sandberg R, Tolias A S. Electrophysiological, transcriptomic and morphologic profiling of single neurons using Patch-seq. Nature Biotechnology 34; 2016:199–203. Two groups report methods for combining electrophysiological patch-clamp recording, morphologic characterization, and single-cell RNA sequencing of neurons in brain slices. Both groups sample several dozen interneurons in the L1/2 layer of the mouse somatosensory cortex. Their methodology involves aspirating the contents of the neuronal soma into the same micropipette that is used for the patch-clamp recording. The aspirated material is then expelled for reverse transcription and sequence analysis in an RNA-Seq protocol. This procedure identifies fewer transcripts than conventional RNA-Seq, but it provides information about the expression of several thousand genes, including neurotransmitter receptors and channels. Both groups are able to correlate patterns of gene expression with neuronal function using this “Patch-seq” approach and believe that the methodology will help establish the molecular basis for neural function and the specialization of neuron subtypes.
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Article Watch: July 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, MCG-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.
DNA SEQUENCING AND GENOTYPING
Quick J, Loman N J, Duraffour S, Simpson J T, Severi E, Cowley L, Bore J A, Koundouno R, Dudas G, Mikhail A, Ou´edraogo N, Afrough B, Bah A, Baum J H J, Becker-Ziaja B, Boettcher J P, Cabeza´ Carter L L, Cabrerizo M, Camino-S´a nchez A, Doerrbecker J, Enkirch T, Dorival I G, Hetzelt N, Hinzmann J, Holm T, Kafetzopoulou L E, Koropogui M, Kosgey A, Kuisma E, Logue C H, Mazzarelli A, Meisel S, Mertens M, Michel J, Ngabo D, Nitzsche K, Pallasch E, Patrono L V, Portmann J, Repits J G, Rickett N Y, Sachse A, Singethan K, Vitoriano I, Yemanaberhan R L, Zekeng E G, Racine T, Bello A, Sall A A, Faye O, Faye O, Magassouba N F, Williams C V, Amburgey V, Winona L, Davis E, Gerlach J, Washington F, Monteil V, Jourdain M, Bererd M, Camara A, Somlare H, Camara A, Gerard M, Bado G, Baillet B, Delaune D, Nebie K Y, Diarra A, Savane Y, Pallawo R B, Gutierrez G J, Milhano N, Roger I, Williams C J, Yattara F, Lewandowski K, Taylor J, Rachwal P, J. Turner D, Pollakis G, Hiscox J A, Matthews D A, Shea M K O, Johnston A M, Wilson D, Hutley E, Smit E, Di Caro A, W¨olfel R, Stoecker K, Fleischmann E, Gabriel M, Weller S A, Koivogui L, Diallo B, Ke¨ıta S, Rambaut A, Formenty P, G¨unther S, Carroll M W. Real-time, portable genome sequencing for Ebola surveillance. Nature 530;2016: 228–232. The recent Ebola epidemic in West Africa presented an opportunity for genome sequencing to contribute to epidemiologic tracking of the infectious agent and monitoring responses to vaccines and treatments. This paper documents the deployment of field stations in local, resource-poor locations to perform genomic surveillance of the Ebola virus. DNA sequencing was performed with the MinION
doi: 10.7171/jbt.16-2702-005
instrument from Oxford Nanopore Technologies (Oxford, United Kingdom), a hand-held device weighing ,100 g that monitors sequence-dependent fluctuations in electrical current as a DNA strand passes through a protein nanopore. The principle of operation enables long sequence reads, although its low accuracy requires consensus averaging of many reads to achieve adequate sequence accuracy. The authors transported to West Africa 3 MinION sequencers, 4 laptop computers, a thermocycler, heat-block, pipettes, and sufficient reagents and consumables to perform sequencing. The entire package weighed ,50 kg. They started sequencing within 2 d of arrival in Guinea. The genome sequencing workflow could be accomplished in ,24 h, although the protocol was usually performed over 2 d. Sufficient reads were often accomplished in 1 h of sequencing time. The authors sequenced 142 samples over 148 MinION runs during a 6 mo period and were able to document the lineage history of Ebola virus in the area. Their main logistic obstacles related to lack of continuity in main electrical supplies, which affected the thermocyclers but not the MinION, and difficulties with internet connectivity, which was needed for data analysis. The results of this study validate the practicality of genome sequencing in resource-limited settings for monitoring outbreaks of infectious disease. Lehmann-Werman R, Neiman D, Zemmour H, Moss J, Magenheim J, Vaknin-Dembinsky A, Rubertsson S, Nellg˚a rd B, Blennow K, Zetterberg H, Spalding K, Haller M J, Wasserfall C H, Schatz D A, Greenbaum C J, Dorrell C, Grompe M, Zick A, Hubert A, Maoz M, Fendrich V, Bartsch D K, Golan T, Ben Sasson S A, Zamir G, Razin A, Cedar H, Shapiro A M J, Glaser B, Shemer R, Dor Y. Identification of tissue-specific cell death using methylation patterns of circulating DNA. Proceedings of the National Academy of Sciences USA 113;2016: E1826–E1834. Lehmann-Werman et al. here demonstrate proof of principal that cell-free, circulating DNA released from dying Journal of Biomolecular Techniques 27:84–89 © 2016 ABRF
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cells may be used to identify the tissue of DNA origin. Their methodology relies on detection of cell type-specific methylation markers. Serum or plasma from donors is treated with bisulfite, PCR amplified, and sequenced to quantify epigenetic markers of interest. The authors identify pancreatic b-cell DNA in patients with recently diagnosed type 1 diabetes and in recipients of islet grafts; oligodendrocyte DNA in patients with relapsing multiple sclerosis; neuronal or glial DNA in patients who have recently suffered traumatic brain injury or cardiac arrest; and exocrine pancreatic DNA in patients with pancreatitis or pancreatic cancer. As levels of cell-free DNA from affected tissues are low in the early stages of disease, the methodology is unlikely to contribute to diagnosis for reasons related to detection sensitivity but is hoped to contribute to an understanding of normal tissue dynamics and to provide an alternative approach to monitoring disease progression and treatment efficacy. MOLECULAR SYNTHESIS AND SYNTHETIC BIOLOGY
Hutchison C A, Chuang R-Y, Noskov V N, AssadGarcia N, Deerinck T J, Ellisman M H, Gill J, Kannan K, Karas B J, Ma L, Pelletier J F, Qi Z-Q, Richter R A, Strychalski E A, Sun L, Suzuki Y, Tsvetanova B, Wise K S, Smith H O, Glass J I, Merryman C, Gibson D G, Venter J C. Design and synthesis of a minimal bacterial genome. Science 351;2016:aad6253. This paper is a report on progress at the J. Craig Venter Institute (JCVI) toward defining the minimal, fully synthetic genome needed to specify an autonomously replicating cell. Previously, the group accomplished total chemical synthesis of the 1079 kilobase pair (kbp) genome of Mycoplasma mycoides, which encodes 901 proteins, and introduced it into cells of a different mycoplasma species, Mycoplasma capricolum, under conditions in which the genome of M. capricolum is lost, and only the transplanted genome remains. The resulting strain, JCVI-syn1.0, is subjected to genome minimization in this paper. The result is a strain called JCVI-syn3.0 that has a genome of just 531 kbp encoding 473 proteins and 35 annotated RNAs. Initial attempts to design the minimal genome de novo based on available knowledge of molecular biology failed to produce viable cells, suggesting that current knowledge is incomplete. An alternative approach was to divide up the JCVIsyn1.0 genome into 8 independent modules, delete pieces of each or disrupt function by transposon insertion, and then reassemble the genome for testing cell viability. This approach succeeded in producing a strain with doubling time as rapid as 3 h—a finding of interest, as there is a tradeJOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 27, ISSUE 2, JULY 2016
off between genome size and growth rate in small genomes subject to minimization. Most of the genes that remain included are involved in gene expression, membrane biogenesis, and metabolism and to a lesser extent, genome replication and preservation of genomic information. Most intriguingly, 79 genes (17%) remain unassigned to a category, and 149 are of unknown function. Investigation of these genes may reveal new cellular processes. The authors’ line of synthetic biology research is of fundamental interest in cell and molecular biology. Spin-off methods may prove useful in designing genomes for industrial biosynthesis and therapeutic intervention. MASS SPECTROMETRY
Riley N M, Mullen C, Weisbrod C R, Sharma S, Senko M W, Zabrouskov V, Westphall M S, Syka J E P, Coon J J. Enhanced dissociation of intact proteins with high capacity electron transfer dissociation. Journal of The American Society for Mass Spectrometry 27;2015:520–531. Electron transfer dissociation (ETD) is a method with favorable characteristics for acquiring amino acid sequence information from intact proteins, as it works well with multiply charged precursor ions and minimizes cleavage of labile side chains and post-translational modifications. The implementation of intact protein ETD in ion-trapping instruments, however, is impeded by the limited charge capacity of ion traps, because as the size of the protein precursor increases, its charge state also increases, and the number of precursor ions that can be stored diminishes proportionately. At the same time, product ion spectra become more complex, and signal is split between increasing numbers of fragments. Riley et al. here describe modifications to the newest generation of quadrupole Orbitrap linear ion trap mass spectrometers, the Orbitrap Fusion Lumos (Thermo Fisher Scientific, San Jose, CA, USA). The modifications increase the instrument’s precursor-trapping capacity for use in analysis of intact proteins. As in previous versions of the instrument, precursors are sequestered in the dual-cell quadrupole linear ion trap mass-to-charge ratio analyzer (A-QLT) before reaction with ETD reagent anions, but the location within the A-QLT, where the ions are stored, is altered here to permit storage of larger numbers of precursor cations. This enables enhancement of product ion signal-to-noise ratios for a given acquisition time, with attendant higher-sequence coverage and less need for signal averaging. The modifications are now available on a commercial platform for analysis of intact proteins and top-down proteomics. 85
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Fukuyama Y, Nakajima C, Izumi S, Tanaka K. Membrane protein analyses using alkylated trihydroxyacetophenone (ATHAP) as a MALDI matrix. Analytical Chemistry 88;2016:1688–1695. This work is an extension of previous studies describing a new matrix for matrix-assisted laser desorption/ ionization (MALDI)-mass spectrometry analysis of hydrophobic peptides from membrane proteins: (2,4,6trihydroxyphenyl)octan-1-one (ATHAP), an alkylated version of trihydroxyacetophenone that incorporates an alkyl chain. which confers increased affinity for hydrophobic peptides. The present report extends the use of ATHAP to detection of intact molecular ions of bacteriorhodopsin, a protein with 7 transmembrane domains that is difficult to detect using a-cyano-4-hydroxycinnamic acid or sinapinic acid matrices. ATHAP also supports detection of peptides containing hydrophobic sequences from a variety of other membrane proteins, thereby confirming its general suitability for their analysis. McMillen C L, Wright P M, Cassady C J. Negative ion in-source decay matrix-assisted laser desorption/ ionization mass spectrometry for sequencing acidic peptides. Journal of The American Society for Mass Spectrometry 27;2016:847–855. McMillen et al. study conditions for MALDI in-source decay to acquire amino acid sequence information for deprotonated acidic peptides in negative ion mode. They test 8 acidic peptides ranging from 11 to 33 residues with each of 7 different matrices and identify 1,5-diaminonaphthalene as the matrix supporting the best fragmentation. As in ETD, electron capture dissociation, and MALDI in-source decay in the positive ion mode, product ions resulting from cleavage of the N-Ca bonds predominate. Negative ion in-source decay is expected to be a generally useful method for analysis of acidic peptides. METABOLOMICS
Hensley Christopher T, Faubert B, Yuan Q, LevCohain N, Jin E, Kim J, Jiang L, Ko B, Skelton R, Loudat L, Wodzak M, Klimko C, McMillan E, Butt Y, Ni M, Oliver D, Torrealba J, Malloy Craig R, Kernstine K, Lenkinski Robert E, DeBerardinis Ralph J. Metabolic heterogeneity in human lung tumors. Cell 164;2016:681–694. The authors here combine metabolic labeling with [U-13C]glucose and clinical imaging by dynamic contrastenhanced magnetic resonance imagery (DCE-MRI) and 86
fluoro-2-deoxyglucose positron emission tomography (FDG-PET) to assess metabolic heterogeneity within and between non-small cell lung cancers in human patients undergoing tumor resection. Metabolic labeling provides information about the metabolic fuel(s) preferred by the malignant tissue, DCE-MRI measures tumor perfusion, and FDG-PET shows cellular uptake of glucose. Compared with normal lung tissue, the tumors display both enhanced glycolysis and enhanced tricarboxylic acid cycle (TCA) activity. This is different from the switch from oxidative to glycolytic metabolism characteristic of the Warburg effect. The enhanced glucose uptake shown by FDGPET apparently supports up-regulated flux through both pathways. The authors observe that entry of 13C into the TCA cycle is dominated by pyruvate dehydrogenase. They also detect low enrichment of 13C in acetyl CoA, suggesting that the tumor cells are oxidizing nutrients other than glucose—probably lactate. Metabolic variability in 13C enrichment is observed and is closely correlated with local perfusion. Limited perfusion is linked to high 13C enrichment, suggesting that cells increase their reliance on glucose compared with other substrates. These results illustrate how imaging combined with metabolic labeling can be used to characterize metabolic heterogeneity in tumors. 18
PROTEINS—PURIFICATION AND CHARACTERIZATION
Gault J, Donlan J A C, Liko I, Hopper J T S, Gupta K, Housden N G, Struwe W B, Marty M T, Mize T, Bechara C, Zhu Y, Wu B, Kleanthous C, Belov M, Damoc E, Makarov A, Robinson C V. Highresolution mass spectrometry of small molecules bound to membrane proteins. Nature Methods 13; 2016:333–336. Gault et al. here describe methods for mass spectrometric analysis of intact membrane proteins in which a modified Orbitrap mass spectrometer provides sufficient resolution to discriminate the binding of lipids, detergents, peptides, and drugs. Protein complexes are protected by micelles during translation into the gas phase. Micelles are removed by collisional activation in the higher-energy collisional dissociation cell or in the source region or both. Voltage gradients are chosen to control the release of the protein without disrupting its stoichiometry or interaction with ligands. The authors characterize a variety of membrane proteins, ranging from 26 to 186 kDa. They measure protein–ligand association constants, deconvolute concomitant binding of drug and lipid molecules, and resolve bound lipids from the same class but having different chain length. The direct identification of bound JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 27, ISSUE 2, JULY 2016
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lipids in this way represents a significant advance over existing mass spectrometric methods. Ayyer K, Yefanov O M, Oberth¨ur D, RoyChowdhury S, Galli L, Mariani V, Basu S, Coe J, Conrad C E, Fromme R, Schaffer A, D¨orner K, James D, Kupitz C, Metz M, Nelson G, Xavier P L, Beyerlein K R, Schmidt M, Sarrou I, Spence J C H, Weierstall U, White T A, Yang J-H, Zhao Y, Liang M, Aquila A, Hunter M S, Robinson J S, Koglin J E, Boutet S, Fromme P, Barty A, Chapman H N. Macromolecular diffractive imaging using imperfect crystals. Nature 530;2016:202–206. The sharp, regularly spaced intensity peaks that arise when X-rays are diffracted by the molecules in a wellordered crystal lattice result from constructive interference between the diffracted rays. The resolution at which molecular structure can be reconstructed depends on the Bragg diffraction limit, which is determined, in part, by the regularity of molecular spacing in the crystal lattice. Disorder decreases the intensity of the sharp Bragg intensity peaks and produces continuous modulation of these diffraction intensities. This gives the appearance of light and shadowy regions in the diffraction pattern, an effect seen all too often from imperfect protein crystals. Ayyer et al. have found a way to use this continuous modulation to enhance the structural information obtained by X-ray diffraction. By treating the continuous diffraction pattern as the incoherent sum of diffraction from rigid individual molecules aligned along several discrete crystallographic orientations, they derive more information than the sharp Bragg intensities provide alone. They first use the Bragg intensities to calculate an electron density map at low resolution. Then, they use the continuous diffraction pattern to refine the map to a resolution beyond the Bragg diffraction limit. In this way, they achieve an improvement in the resolution attained for the photosystem II dimer, a very large membrane protein of molecular weight 700 kDa, ˚ It is hoped that the approach will be from 4.5 to 3.5 A. generally applicable for enhancing resolution from macromolecules that form crystals of imperfect quality. PROTEOMICS
Hart-Smith G, Yagoub D, Tay A P, Pickford R, Wilkins M R. Large scale mass spectrometry-based identifications of enzyme-mediated protein methylation are subject to high false discovery rates. Molecular and Cellular Proteomics 15;2016:989–1006. Special functional significance has been attributed to the post-translational methylation of histones. However, JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 27, ISSUE 2, JULY 2016
methylation occurs widely among proteins, and the location of methylation sites (most commonly on lysine and arginine residues) is of more general interest. Hart-Smith et al. here illustrate how to perform rigorous validation of methylation sites through heavy methyl stable isotope labeling in cell culture. In studies of yeast cells, they provide 13CD3-labeled methionine in the culture medium. Methionine is the precursor of S-adenosyl-L-methionine, the methyl donor used by all known methyltransferases. The resulting mass shifts of methylated peptides allow the number of incorporated methyl groups to be counted. Sources of artifactual methylation include the esterification of glutamic and aspartic acids during sample preparation protocols that use methanol or isopropanol. These reactions may be mistaken for enzyme-mediated monoor dimethylation of proximal arginine or lysine residues during automated spectrum-matching searches, but are distinguished from cellular methylation by the labeling procedure. The authors show that false discovery rates estimated by targetdecoy approaches greatly underestimate actual rates of false discovery of methylation sites. This effect is attributed to the large number of amino acid combinations capable of producing peptide sequences that are isobaric with methylated peptides of different sequence. The rate of false discovery remains high even when antibody-based purification of methylated peptides is used. Therefore, the authors caution against reliance upon target-decoy estimates in studies of protein methylation. Moore S M, Hess S M, Jorgenson J W. Extraction, Enrichment, solubilization, and digestion techniques for membrane proteomics. Journal of Proteome Research 15;2016:1243–1252. This paper revisits the optimal conditions for preparing membrane proteins for proteomic analysis. In studying yeast cells, the authors see optimal numbers of identified proteins, best quantitative recovery of most proteins, and greatest convenience in a protocol that incorporates cell disruption in a French press, no further enrichment step, solubilization with 1% sodium deoxycholate while applying sonication, and digestion with trypsin at 37°C. Cell lysis by sonication, cryopulverization, freeze/thawing, or bead beating provided no advantage. Enrichment by chloroform/methanol partitioning or acetone precipitation resulted in loss of some proteins. Solubilization with methanol and use of enzymes other than trypsin were also not advantageous. FUNCTIONAL GENOMICS AND PROTEOMICS
Kleinstiver B P, Pattanayak V, Prew M S, Tsai S Q, Nguyen N T, Zheng Z, Joung J K. High-fidelity CRISPR–Cas9 nucleases with no detectable genomewide off-target effects. Nature 529;2016:490–495. 87
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Slaymaker I M, Gao L, Zetsche B, Scott D A, Yan W X, Zhang F. Rationally engineered Cas9 nucleases with improved specificity. Science 351;2016:84–88. Genome editing with the clustered, regularly interspaced, short palindromic repeat (CRISPR)-associated 9 (Cas9) protein, used in conjunction with a single-guide RNA (sgRNA), relies on double-stranded DNA cleavage at a site with base sequence complementary to the sgRNA. Cas9 shows high-sequence specificity of target cleavage, even in mammalian genomes, which are some 18003 larger than the bacterial genomes in which the Cas9 protein originally evolved to excise viral DNA. Nevertheless, nonspecific binding by Cas9 is measureable, and off-target cleavage occurs occasionally. The engineering of Cas9 with improved sequence selectivity is now an important goal, as Cas9-based gene editing for clinical purposes is within view. Two groups here describe improvements in Cas9 specificity. Both groups exploit knowledge that the affinity of binding between Cas9-sgRNA and its target has a sequence-specific component and a sequence-nonspecific component that results from interactions between protein and DNA backbone. Both groups approach specificity enhancement by mutating amino acid residues contributing to the nonspecific component of the total binding affinity. With the use of Cas9 from Streptococcus pyrogenes, Kleinstiver et al. mutate residues that contact the backbone of the DNA target strand, whereas Slaymaker et al. mutate residues that contact the complementary DNA single strand. Although overall binding affinity is weakened in both approaches, the difference between onand off-target affinity is broadened. Both groups show that the overall weakening is not so great as to impair on-target nuclease activity significantly, but they demonstrate improved specificity by unbiased detection of cleavage using the most sensitive available high-throughput sequencing methods. The new proteins are expected to speed genome-editing applications in basic science and hasten clinical application. Rajagopal N, Srinivasan S, Kooshesh K, Guo Y, Edwards M D, Banerjee B, Syed T, Emons B J M, Gifford D K, Sherwood R I. High-throughput mapping of regulatory DNA. Nature Biotechnology 34;2016:167–174. Korkmaz G, Lopes R, Ugalde A P, Nevedomskaya E, Han R, Myacheva K, Zwart W, Elkon R, Agami R. Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9. Nature Biotechnology 34;2016:192–198. The human genome is believed to contain .500,000 enhancers (DNA sites at which transcription factors bind 88
and regulate the transcription of sometimes quite-distantly located genes through chromatin looping). The need to grapple with the phenomenal complexity that this number of enhancers represents is highlighted by ongoing reports of their involvement in diseases, such as cancer and autoimmunity, and the therapeutic potential of interfering with the function of proteins, including transcription factors, with which they interact. Existing high-throughput methods for analyzing enhancers involve cloning regulatory elements into plasmid vectors, but this approach does not capture the function of enhancers in their native genomic context. The present 2 papers initiate the use of the CRISPR-Cas9 system to conduct high-throughput mapping of regulatory elements at single-base resolution in the native context. Rajagopal et al. study the regulatory elements that control expression of 4 embryonic stem cell-specific genes, identify proximal and distal regulatory elements necessary for their expression, and discover regulatory elements unmarked by typical epigenetic or chromatin features. Korkmaz et al. describe 2 genetic screens to identify enhancer elements that bind TP53 (tumor protein 53) and estrogen receptor a, identify several endogenous enhancer elements, and characterize domains essential for their activity. These studies represent a new milestone in highthroughput functional screening of regulatory elements in their native context. Kline M C, Romsos E L, Duewer D L. Evaluating Digital PCR for the quantification of human genomic DNA: accessible amplifiable targets. Analytical Chemistry 88;2016:2132–2139. Digital PCR (dPCR) is a method for measuring the quantity of a particular DNA target sequence in a given sample. The sample is partitioned into small volumes (chambers or equal-sized droplets) so that a given volume contains 0 to a few target sequences. The volumes are subjected to a predetermined number of PCR amplification cycles and then scored for the presence or absence of fluorescence signal from the target. The number of volumes that contain at least 1 target sequence relative to the total number of volumes scored is described by the Poisson distribution and provides a measure of the concentration of target sequences in the original sample. The measurement does not depend on comparison with a DNA quantification standard. Rather, it can be used to create reference standards as calibrants for a variety of applications. Kline et al. here report a source of bias in dPCR measurements. They show that dPCR measures not the absolute number of targets in a sample but rather, the number of targets that are accessible and amenable to amplification. Untreated DNA is often treated with endonuclease to render targets accessible, but nucleases can destroy, as well as reveal, targets. If nucleases JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 27, ISSUE 2, JULY 2016
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are to be used, the fraction of targets they make inaccessible must be measured independently. Nucleases giving mean fragment lengths of several thousand base pairs are shown to do less damage than those that cut more frequently. When estimating the amount of human genomic DNA, several different targets should be measured, and the results obtained with different dPCR platforms (chamber and droplet) should be compared to check for agreement. Accuracy further depends on assessment of chamber or droplet volume, which is presently difficult to achieve. These results and recommendations will be of interest to all investigators using dPCR in their research.
hydrochloride (Benadryl), lidocaine hydrochloride, diazepam (Valium), and fluoxetine hydrochloride (Prozac). All drug products are formulated to meet standards set forth by U.S. Pharmacopeia. The formulations are concentrated aqueous or alcohol-based solutions ready for dilution when needed. Solid formulations, such as tablets, however, would require modules for drying, powder transport, solids blending, and tableting that are still under development. With appropriate U.S. Food and Drug Administration approvals, the technology incorporated into the instrumentation described in this paper is expected to enable local production of medications for hospitals, healthcare organizations, pharmaceutical development, and humanitarian aid.
DRUG DESIGN AND CHARACTERIZATION
Adamo A, Beingessner R L, Behnam M, Chen J, Jamison T F, Jensen K F, Monbaliu J-C M, Myerson A S, Revalor E M, Snead D R, Stelzer T, Weeranoppanant N, Wong S Y, Zhang P. Ondemand continuous-flow production of pharmaceuticals in a compact, reconfigurable system. Science 352;2016:61-67. Drug companies generally make pharmaceuticals in large batches using procedures that require large manufacturing plants, protracted periods of time, and extensive storage facilities. Drug shortages are often the result of limitations inherent in such batch-wise manufacturing. The authors of the present paper have developed a continuous manufacturing platform that combines synthesis and drug product formulation into a single unit the size of a refrigerator. It uses continuous flow to streamline, automate, and integrate synthesis processes at the grams/hour scale. The instrument is comprised of reconfigurable modules: an upstream unit houses feeds, pumps, reactors, separators, and pressure regulators for chemical synthesis of an active pharmaceutical ingredient (API) from simple chemical precursors, and a downstream unit contains precipitation tanks, crystallizers, and filters for purification and formulation of the drug product. Elevated pressures and temperatures are used to accelerate reactions, and reagent concentrations are kept high to minimize waste production. An in-line attenuated total reflection Fourier transform infrared spectrometer provides realtime monitoring of the APIs formed. The modules are reconfigured to accomplish synthesis of 4 products encompassing a range of chemical complexity: diphenhydramine
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CELL BIOLOGY AND TISSUE ENGINEERING
Fuzik J, Zeisel A, Mate Z, Calvigioni D, Yanagawa Y, Szabo G, Linnarsson S, Harkany T. Integration of electrophysiological recordings with single-cell RNA-seq data identifies neuronal subtypes. Nature Biotechnology 34;2016:175–183. Cadwell C R, Palasantza A, Jiang X, Berens P, Deng Q, Yilmaz M, Reimer J, Shen S, Bethge M, Tolias K F, Sandberg R, Tolias A S. Electrophysiological, transcriptomic and morphologic profiling of single neurons using Patch-seq. Nature Biotechnology 34; 2016:199–203. Two groups report methods for combining electrophysiological patch-clamp recording, morphologic characterization, and single-cell RNA sequencing of neurons in brain slices. Both groups sample several dozen interneurons in the L1/2 layer of the mouse somatosensory cortex. Their methodology involves aspirating the contents of the neuronal soma into the same micropipette that is used for the patch-clamp recording. The aspirated material is then expelled for reverse transcription and sequence analysis in an RNA-Seq protocol. This procedure identifies fewer transcripts than conventional RNA-Seq, but it provides information about the expression of several thousand genes, including neurotransmitter receptors and channels. Both groups are able to correlate patterns of gene expression with neuronal function using this “Patch-seq” approach and believe that the methodology will help establish the molecular basis for neural function and the specialization of neuron subtypes.
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