ARTICLE WATCH

Article Watch: September 2017 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

Huddleston J, Chaisson M J, Steinberg K M, Warren W, Hoekzema K, Gordon D, Graves-Lindsay T A, Munson K M, Kronenberg Z N, Vives L, Peluso P, Boitano M, Chin C-S, Korlach J, Wilson R K, Eichler E E. Discovery and genotyping of structural variation from long-read haploid genome sequence data. Genome Research 27;2017:677–685. The ability to detect single-nucleotide variants in the euchromatic portion of the human genome is very good, but detection of structural variants, including insertions, deletions, and inversions of $50 bp, and indels of 1–49 bp, is more difficult because of the stronger association of such variants with repetitive sequences. The present paper reports de novo construction of datasets describing haplotyperesolved (phased) human genetic variation using long-read sequencing. The authors use sequencing technology from Pacific Biosciences (Menlo Park, CA, USA) and apply it to 2 haploid human genomes. The haploid genomes are derived from hydatidiform moles. Such moles arise either by fertilization of an anucleated egg by sperm or by loss of the maternal complement of chromosomes after fertilization. Compared with short-read sequence data, the authors estimate that long-read sequencing provides a 5-fold increase in detection sensitivity for structural variants and indels of 7–1000 bp in length. This advantage derives mostly from an improved ability to map the longer reads in repeat-rich, high G-C content and low-complexity DNA by anchoring alignments within the flanks of such regions. The authors simulate a single diploid genome from their 2 haploid datasets and show that the ability to call heterozygous structural variants is improved by resolving the haplotypes first, even with short-read sequence data. This finding indicates a general advantage to uncoupling variant discovery from genotyping.

doi: 10.7171/jbt.17-2803-005

Journal of Biomolecular Techniques 28:127–134 © 2017 ABRF

Weisenfeld N I, Kumar V, Shah P, Church D M, Jaffe D B. Direct determination of diploid genome sequences. Genome Research 27;2017:757–767. Weisenfeld et al. here test a previously described methodology (Zheng GX. et al. Haplotyping germline and cancer genomes with high-throughput linked-read sequencing. Nature Biotechnology 34;2016:303–311) for de novo assembly of phased sequences from short-read data acquired with diploid cells. The methodology uses severalmillion gel beads, each of which contains many copies of a 16-base barcode unique to that bead. The beads are distributed individually among ;1 million oil droplets in a microfluidic device. High molecular weight DNA and enzymatic reagents are added to the droplets so that each droplet receives several long DNA molecules. The gel beads are then dissolved. This initiates whole-genome primer extension. The resulting constructs consist of a barcode, along with ;350 bp of genomic DNA sandwiched between Illumina adapters (Illumina, San Diego, CA, USA). The barcode is located at the beginning of the first read in a pair. The constructs are pooled and sequenced on an Illumina instrument. Groups of read-pairs are identified by barcode: the probability of sequences derived from different haplotypes having the same barcode is very small. The process requires only 1 ng genomic DNA. Microfluidics and assembly software for this procedure are provided commercially by 10X Genomics (Pleasanton, CA, USA). The authors process 7 human samples in this way, including 4 for which parental data were available so that the accuracy of phasing could be established. The methodology yields phase blocks much longer than previous diploid assemblies and avoids bias introduced by reference sequences. CARBOHYDRATES AND GLYCOPROTEINS

Kalxdorf M, Gade S, Eberl H C, Bantscheff M. Monitoring cell-surface N-glycoproteome dynamics by quantitative proteomics reveals mechanistic insights into macrophage differentiation. Molecular & Cellular Proteomics 16;2017:770–785.

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Kalxdorf et al. describe methodology for improved proteomic sampling of plasma membrane proteins. The methodology is based chiefly on enrichment of sialylated proteins. The authors oxidize glycans with sodium metaperiodate and then label with biotin by reacting the resulting aldehyde groups with alkoxylamine-PEG4-biotin in the presence of aniline as a catalyst. Biotinylated proteins are then enriched on streptavidin-coated beads. Bound proteins are trypsinized, and the resulting peptide mixture is analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The procedure enabled an average of 835 different plasma membrane proteins to be identified on each member of a panel of standard laboratory cell lines and primary cells. The authors document marked differences in surface proteome composition between lymphoid primary cells and corresponding cell lines of similar origin, presumably reflecting selection for high growth rate in culture, among other factors. The authors note that these differences may contribute to observed variation in drug sensitivity and advocate the testing of candidate drugs on primary cells wherever possible. The authors proceed to characterize changes in plasma membrane proteome that occur during differentiation of THP-1 monocytes to macrophages. With the use of time-resolved labeling by tandem mass tagging, they distinguish the rates of several processes that occur during the transition, including secretion of collagens and other extracellular matrix proteins, relocalization of proteins stored in intracellular compartments to the plasma membrane, internalization or degradation of proteins no longer needed on the cell surface, and presentation of newly synthesized proteins. They correlate these changes with changes in cell morphology and behavior. Finally, marked perturbation of the macrophage plasma membrane proteome by the tyrosine kinase inhibitor dasatinib is documented. This results from offtarget interactions. The observations imply impairment of macrophages differentiation by dasatinib. The methodology sets a new standard for biomedical studies of the plasma membrane proteome. METABOLOMICS

Leeming M G, Donald W A, O’Hair R A. Nontargeted identification of reactive metabolite protein adducts. Analytical Chemistry 89;2017:5748–5756. Some xenobiotic substances (drugs, insecticides, industrial chemicals, toxins, etc.) are metabolically converted to reactive species that covalently bind to cell constituents. For example, acetaminophen [N-acetyl-p-aminophenol (APAP)] is converted to the reactive metabolite N-acetylp-benzoquinone imine (NAPQI), which contributes to 128

toxicity in acetaminophen overdose. Leeming et al. describe a nontargeted method for identifying macromolecules that become modified by such reactive metabolites. To illustrate the method, they incubate rat liver microsomes with a mixture of unlabeled and [13C]6-labeled APAP. With the use of LC-MS/MS analysis, they are able to identify tryptic peptides resulting from APAP adduction by the pairs of isotopic signals that result. The method does not require metabolite trapping or foreknowledge of the chemical structure of adducts or the identity of targeted amino acid residues. Subsequent, specifically parameterized database searches reveal further, similar, residue modifications. The approach reveals previously unknown protein targets of NAPQI modification and is anticipated to be useful for studying mechanisms of toxicity of many compounds. MASS SPECTROMETRY

Fenner M A, Chakrabarty S, Wang B, Pagnotti V S, Hoang K, Trimpin S, McEwen C N. An LC/MS method providing improved sensitivity: electrospray ionization inlet. Analytical Chemistry 89; 2017:4798–4802. Solvent-assisted ionization (SAI) is an inlet ionization method in which a liquid stream containing dissolved analytes is introduced directly into the heated inlet transfer tube connecting the atmospheric pressure region of a mass spectrometer source to the vacuum region of the instrument. Ionization is believed to occur within the transfer tube as a result of heating and the high velocity of air passing through the transfer tube. In one variant of SAI, termed electrospray ionization inlet (ESII), the ionization process is assisted by the placement of an electrical charge on the tip of the fused silica tube that introduces the liquid stream into the mass spectrometer inlet tube. The ionization process that results differs from true electrospray ionization (ESI) in that it does not require a threshold voltage to induce formation of a Taylor cone from the liquid stream. The voltages required for ESII are actually lower than ESI threshold voltages; indeed, even if the voltage is reduced to 0, SAI would still occur. In the present paper, Fenner et al. report experiments in which LC-ESII/MS provided a significant improvement in sensitivity compared with LC-ESI/MS. They supply ESII voltage via a metal union forming a junction in the LC eluent stream. They test a mixture of drugs, a peptide standard mixture, and a protein digest, and observe substantially higher ion abundance and lower chemical background in all cases at flow rates of 15–55 mL/min. Su X, Lu W, Rabinowitz J D. Metabolite spectral accuracy on Orbitraps. Analytical Chemistry 89; 2017:5940–5948. JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 3, SEPTEMBER 2017

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This paper alerts mass spectrometrists to a systematic source of error in measuring the abundance of isotopic peaks with Orbitrap mass spectrometers. The inaccuracy manifests as undermeasurement of the abundance of heavier isotopologues. Accurate measurement of the fractional abundance of isotopologues (termed “spectral accuracy”) is critical in certain applications. For example, in the measurement of metabolic flux with stable isotope tracers, even small errors in spectral accuracy may result in large errors in flux measurement and lead to incorrect conclusions. The magnitude of the error is found to increase with mass above the lower end of the scanned mass range. For example, when scanning from mass-to-charge ratio (m/z) 75–1000, it results in 20% undermeasurement of (M + 1)/ (M + 0) at m/z 600. The effect is not caused by space charging as a result of trap overfilling. This too can produce systematic errors in spectral accuracy, but space-charging errors can be reduced by decreasing the instrument’s automatic gain control setting, which regulates the number of ions introduced into the trap. The present error could not be eliminated in this way. It could be reduced, however, by decreasing resolving power, which shortens the residence time of ions in the Orbitrap analyzer during mass measurement. The authors reason that the present inaccuracy may result from weaker containment of high-mass ions in the C-trap, which would lead to injection of ions into the Orbitrap analyzer with suboptimal trajectories. They argue that in this context, the time-dependent loss of signal may disproportionately affect lower-abundance isotopologs and suggest possible mechanisms for the effect. The authors resolve the problem by splitting the scanned mass range: for example, when collecting data from m/z 75–1000, they scan a lower m/z range (75–500) and a higher m/z range (500–1000) separately. An abundance correction algorithm of improved accuracy is also provided. Hughes C S, Zhu C, Spicer V, Krokhin O V, Morin G B. Evaluating the characteristics of reporter ion signal acquired in the Orbitrap analyzer for isobaric mass tag proteome quantification experiments. Journal of Proteome Research 16;2017:1831–1838. This paper follows up an apparent artifact in the distribution of signal intensities acquired with Orbitrap mass analyzers. During a quantitative proteomic comparison of 2 cell types using 6-Plex Tandem Mass Tags, the authors acquired reporter ion signal intensity data in the MS3 mode. The protocol involves isolating a product ion formed by collision-induced dissociation of a labeled peptide precursor ion and then subjecting this product to another round of collision-induced dissociation at higher energy and measuring the resulting MS3 reporter fragment JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 3, SEPTEMBER 2017

intensities. The procedure is performed to avoid signal ratio compression resulting from coisolation of precursors. When the authors plotted the signal intensities for a given reporter ion, they expected to see a smooth distribution of intensity values reflecting the range of abundance of the peptides being quantified. Instead, they observed a discontinuity in the intensity data. There was an absence of signals over a circumscribed interval of signal intensity near the lower end of an otherwise smooth distribution. Retrospective analysis of other isobaric tagging datasets suggests that this artifact is widespread and is attributable to Orbitrap analyzers. The cause of the artifact remains unidentified. Its relationship (if any) to the effect observed by Su et al. (see above) is unknown. Mass spectrometrists will want to be mindful of these idiosyncratic features of data acquired with Orbitrap analyzers. Readers are also referred to a companion paper by the same group (Hughes C S. et al. Investigating acquisition performance on the Orbitrap fusion when using tandem MS/MS/MS scanning with isobaric tags. Journal of Proteome Research 16;2017:1839–1846), which provides a systematic analysis of conditions for acquiring MS3 data in proteomic studies involving isobaric tagging for relative quantification of peptides. PROTEOMICS

˚ Thul P J, Akesson L, Wiking M, Mahdessian D, Geladaki A, Ait Blal H, Alm T, Asplund A, Bj¨ork L, Breckels L M, B¨a ckstr¨om A, Danielsson F, Fagerberg L, Fall J, Gatto L, Gnann C, Hober S, Hjelmare M, Johansson F, Lee S, Lindskog C, Mulder J, Mulvey C M, Nilsson P, Oksvold P, Rockberg J, Schutten R, ˚ Sj¨ostedt E, Skogs M, Schwenk J M, Sivertsson A, Stadler C, Sullivan D P, Tegel H, Winsnes C, Zhang C, Zwahlen M, Mardinoglu A, Pont´en F, von Feilitzen K, Lilley K S, Uhl´en M, Lundberg E. A subcellular map of the human proteome. Science 356;2017:aal3321. This paper announces the compilation of a resource called the Cell Atlas that comprehensively specifies the subcellular distribution of proteins in the human proteome. Various approaches are capable of providing information about subcellular distribution. The authors combine transcriptomics, antibody-based immunofluorescence microscopy, and MS. Firstly, they use RNA sequencing data to identify the genes expressed in each of 56 human cell lines of diverse origin. Secondly, they use a panel of 13,993 affinitypurified antibodies, validated for specificity, to perform immunofluorescence and high-resolution confocal microscopy. Their studies yield information about the distribution of 12,003 proteins among 30 subcellular structures. In this 129

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way, the proteomic composition of 13 major subcellular organelles is defined. Thirdly, the authors combine density gradient cell fractionation with quantitative MS to measure the distribution profiles of 5020 proteins. The datasets are especially interesting, as they identify many proteins that localize to multiple compartments. They also highlight proteins that display variability between individual cells. The Cell Atlas resource represents a gold standard against which to assess the biologic function of individual proteins in mammalian cells. Schr¨a der C U, Lee L, Rey M, Sarpe V, Man P, Sharma S, Zabrouskov V, Larsen B, Schriemer D C. Neprosin, a selective prolyl endoprotease for bottom-up proteomics and histone mapping. Molecular & Cellular Proteomics 16;2017:1162–1171. This paper adds a new reagent enzyme to the proteincleaving armamentarium available for mass spectral analysis in proteomic studies or protein structural analysis. The enzyme, named neprosin, was discovered in secretions of the carnivorous pitcher plant, but it is characterized here as a recombinant enzyme. The enzyme is a novel prolyl endopeptidase that cleaves C-terminal to proline and alanine. However, the number of missed cleavage sites is high, particularly after alanine, for reasons yet to be determined. Several characteristics well suit neprosin for work as a reagent protease: digestion proceeds to a stable end point; the enzyme is active in acidic conditions (pH 2.5); and it cleaves polypeptide substrates of any size. Its specificity is complementary to that of the commonly used enzyme trypsin. The authors demonstrate one instance in which this is a particular advantage. In a single digest, it generates peptide 1–38 of histone H3 and peptide 1–31 of histone H4. These regions, of course, bear post-translational modifications that determine epigenetic interactions. FUNCTIONAL GENOMICS AND PROTEOMICS

Beagrie R A, Scialdone A, Schueler M, Kraemer D C, Chotalia M, Xie S Q, Barbieri M, de Santiago I, Lavitas L-M, Branco M R, Fraser J, Dostie J, Game L, Dillon N, Edwards P A W, Nicodemi M, Pombo A. Complex multienhancer contacts captured by genome architecture mapping. Nature 543;2017: 519–524. Chromatin conformation capture and the methods that have arisen from it, such as 4C and Hi-C, have contributed greatly to knowledge of nuclear architecture. However, these methodologies have limitations that arise from the 130

ligation and digestion processes upon which they depend. For example, they cannot quantify 3-way chromatin contacts accurately, and discrepancies with 3-dimensional (3D) fluorescence in situ hybridization (FISH) sometimes occur. Beagrie et al. here describe a different approach to investigate chromatin organization, called genomic architecture mapping. The new method avoids ligation and provides information that is complementary to conformation capture. Cells are fixed, and the population of nuclei is subjected to cryosectioning at a thickness of 220 nm in random orientations to produce one slide from each nucleus. Each nuclear slice is transferred to a PCR tube by laser-capture microdissection and there, is subjected to DNA extraction, fragmentation, amplification by single-cell whole-genome amplification, and sequencing. Analysis of the data presupposes that sequences of DNA that are closer together in 3D space will be detected more often within the same section. The authors have further developed a statistical model that distinguishes specific interactions from random ones on the basis of departure from expected distribution based simply on linear genomic distance. They use their methodology to study mouse embryonic stem cells, and discover abundant 3-way contacts, especially among regions that are strongly transcribed or contain superenhancers. Particular advantages of the methodology are that it requires only small numbers of cells, can work with tissue sections as well as dispersed cells, and is suitable for describing such higher-order features as radial distribution of chromosomes and regions of chromosome compaction. Significant stimulation of discovery in the field of nuclear architecture is anticipated to result from its use. Kim Y B, Komor A C, Levy J M, Packer M S, Zhao K T, Liu D R. Increasing the genome-targeting scope and precision of base editing with engineered Cas9cytidine deaminase fusions. Nature Biotechnology 35;2017:371–376. Recently, the authors’ laboratory described methodology for inducing programmable, single-nucleotide base changes in the DNA of living cells using clustered regularly interspaced short palindromic repeats (CRISPR) but without inducing double-stranded breaks in DNA, which may, in turn, induce indels. Their procedure is accomplished with a fusion protein containing a catalytically inactive CRISPR-associated protein 9 (Cas9) nuclease (dCas9). This is conjugated with a cytidine deaminase and an inhibitor of base excision repair. The construct converts cytidines to uridines within a 5 nt window, specified by a small guide RNA (sgRNA), and results in conversion of C:G base pairs to T:A base pairs. The present paper reports refinements to this methodology. The authors engineer JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 3, SEPTEMBER 2017

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Cas9 variants that have different sequence specificities for the protospacer-adjacent motif, the DNA sequence immediately following the sequence targeted by Cas9. These variants expand the number of sites that can be targeted for base editing. The authors also engineer the cytidine deaminase domain of their base editor to narrow from 5 to 1–2 nt the width within which base editing occurs. This improves the precision of editing. Klann T S, Black J B, Chellappan M, Safi A, Song L, Hilton I B, Crawford G E, Reddy T E, Gersbach C A. CRISPR-Cas9 epigenome editing enables highthroughput screening for functional regulatory elements in the human genome. Nature Biotechnology 35;2017:561–568. Klann et al. here contribute to the CRISPR-based methodology available for high-throughput screening to identify genomic regulatory elements. Although CRISPRCas9 screens with pooled sgRNA libraries have been used to ablate regulatory elements for this purpose, the approach has been limited both because target sequence requirements preclude true saturation screening and because it cannot support gain-of-function screening, being restricted to lossof-function. The approach adopted by Klann et al. uses a dCas9 fused to protein domains that either stimulate or repress gene expression by modulating epigenomic marks, but without altering DNA sequence. To repress gene expression, they fuse dCas9 to the Kr¨uppel-associated box domain, which recruits factors that trimethylate histone H3 on Lys9, leading to formation of heterochromatin. To stimulate gene expression, they fuse dCas9 to the E1Aassociated protein p300 histone acetyltransferase core domain, which acetylates histone H3 on Lys27, providing opportunities for gain-of-function screening. The authors construct sgRNA libraries that target DNase 1-sensitive sites, as many (although not all) regulatory sequences are DNase 1 sensitive. In proof-of-principle experiments, they study regulatory elements for the b-globin cluster and the HER2 oncogene locus in human cells and identify known and previously unidentified regulatory elements. Responses in these screens are mostly of small magnitude (2 times or less), raising the possibility that the limited assay sensitivity may cause some regulatory sequences to be missed. Nevertheless, the methodology provides a scalable approach to study gene regulation. It has the flexibility to be targeted on the basis of many criteria, not just DNase sensitivity. Possibilities include ChIP-Seq and single-nucleotide polymorphism-based genome-wide association studies. The opportunity to activate as well as suppress genes is expected to provide new information about interactions among genes. JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 3, SEPTEMBER 2017

CELL BIOLOGY AND TISSUE ENGINEERING

Merkle F T, Ghosh S, Kamitaki N, Mitchell J, Avior Y, Mello C, Kashin S, Mekhoubad S, Ilic D, Charlton M, Saphier G, Handsaker R E, Genovese G, Bar S, Benvenisty N, McCarroll S A, Eggan K. Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations. Nature 545;2017:229–233. Prospects for the use of human pluripotent stem cells in regenerative therapies raise concern about the genetic integrity and stability of such cells, whether they are derived from embryos or from induction of pluripotency in differentiated cells. Merkle et al. here report exome sequencing of 140 independent human embryonic stem cell lines and reveal that 5 of these lines contain some cells that carry dominant-negative mutations in the gene encoding the tumor suppressor TP53. The prevalence of such mutant cells within lines containing them increases with time, suggesting that the mutations may confer a selective advantage on the carrier. The authors go on to analyze previously published RNA sequence data from 117 human pluripotent stem cell lines and find another 9 TP53 mutations. The mutation reached 100% prevalence in 3 cell lines. Mutations in other genes were much less common. The presence of such cancer-related genetic change underscores the importance of monitoring pluripotent stem cells and their derivatives by genome-wide analysis at key steps in their use for studies connected with regenerative medicine. Rapid progress is presently occurring in methods for the production and use of organoids in biomedical research. Organoids are self-organizing structures derived in culture from pluripotent stem cells. They contain diverse cell types that mimic the composition and architecture of mature organs. Organoids can be used either in vitro or in vivo to gain knowledge of developmental mechanisms, to test drugs, and to explore potential restorative therapies. Six recent papers dealing with 4 types of organoid are reviewed here. Quadrato G, Nguyen T, Macosko E Z, Sherwood J L, Min Yang S, Berger D R, Maria N, Scholvin J, Goldman M, Kinney J P, Boyden E S, Lichtman J W, Williams Z M, McCarroll S A, Arlotta P. Cell diversity and network dynamics in photosensitive human brain organoids. Nature 545;2017:48–53. Birey F, Andersen J, Makinson C D, Islam S, Wei W, Huber N, Fan H C, Metzler K R, Panagiotakos G, Thom N, O’Rourke N A, Steinmetz L M, Bernstein J A, Hallmayer J, Huguenard J R, Pas¸ca S P. Assembly 131

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of functionally integrated human forebrain spheroids. Nature 545;2017:54–59. Two studies extend capabilities for modeling brain development through organoid culture. Quadrato et al. identify ways to modify existing brain organoid culture conditions to prolong viability for more than 9 mo. Such extended durations allow time for differentiation of component cells into diverse cell types that are related to the classes of cells in neural tissue. Cells similar to the various types comprising the cerebral cortex and retina are among those the authors identify while characterizing gene expression in 80,000 individual cells from 31 human brain organoids by RNA sequencing. Neuronal cells acquired structures like dendritic spines during culture, which have previously been difficult to generate in vitro. The methodology thereby opens opportunities to study developmental processes, such as synaptic pruning. By 8 mo, organoids developed spontaneously active neurons and neural networks. Interestingly, they also develop photosensitive cells that respond to light. Birey et al. also exploit the capabilities of very large-scale, single-cell transcriptome analysis but apply it to study neuronal migrations that occur during brain development. They identify different culture conditions that support the differentiation of human pluripotent stem cells into structures that resemble either the dorsal forebrain (distinguished by the presence of glutamatergic excitatory neurons) or the ventral forebrain (distinguished by the presence of GABAergic interneurons). Upon fusing these 2 types of structure to form forebrain-like organoids, they observe, by live cell imaging, migration of interneurons and their maturation to form functional interactions with glutamatergic neurons. Such migration recapitulates processes that take place during fetal development. Furthermore, when they form ventral forebrain structures with pluripotent cells from patients with Timothy syndrome— a developmental condition in which interneuron migration is abnormal—the interneurons in culture display a cell-autonomous migration defect. Making use of the knowledge that in Timothy syndrome, L-type calcium channels contribute to the migration defect, the authors show that pharmacologic manipulation of these channels rescues the interneuron defect in vitro. These results indicate that brain organoids may be used to study interaction of different types of neurons that would otherwise be inaccessible during development. Chen Y-W, Huang S X, de Carvalho A L, Ho S-H, Islam M N, Volpi S, Notarangelo L D, Ciancanelli M, Casanova J-L, Bhattacharya J, Liang A F, Palermo L M, Porotto M, Moscona A, Snoeck H-W. A 132

three-dimensional model of human lung development and disease from pluripotent stem cells. Nature Cell Biology 19;2017:542–549. Chen et al. have identified conditions that support the development of lung bud organoids from human pluripotent stem cells. RNA sequencing indicates that these organoids contain mesoderm and pulmonary endoderm cells, although they do not form mature club cells, ciliated cells, or basal cells. They do develop into branching airway and early alveolar structures in Matrigel 3D culture or when transplanted into immunodeficient recipient mice. After 6 mo in Matrigel culture, the organoids match gene-expression signatures of second-trimester human embryos. The authors infect their organoids in vitro with respiratory syncytial virus, which causes bronchiolitis with small airway obstruction in children, and show that the virus reproduces morphologic changes of human lung infection, including shedding of infected cells into organoid luminal structures. The authors also conduct experiments to model pulmonary fibrosis. Deletion of the HSP1 gene—deficiency of which is associated with earlyonset pulmonary fibrosis—produces accumulation of extracellular matrix and mesenchymal cells. Therefore, it is hoped that the lung-bud organoids will provide a model to study idiopathic pulmonary fibrosis, a devastating disease in humans. Koehler K R, Nie J, Longworth-Mills E, Liu X-P, Lee J, Holt J R, Hashino E. Generation of inner ear organoids containing functional hair cells from human pluripotent stem cells. Nature Biotechnology 35;2017:583–589. Koehler et al. report continued progress in the development of organoids that mimic the inner ear. The human inner ear is a complex, vulnerable structure. It contains chambers that sense sound, gravity, and angular acceleration by using mechanosensory hair cells to generate electrical signals in response to mechanical stress. By timed manipulation of 4 signaling pathways, the authors previously differentiated mouse pluripotent stem cells in Matrigel culture to produce otic vesicle-like structures with convoluted and multichambered morphologies that resemble the inner ear’s membranous labyrinth. The organoids contain sensory hair cells facing the lumen. In the present paper, the authors document similar success with human pluripotent stem cells. Neurons also arise in these structures, and over a period of 2 mo in culture—about the same period during which development occurs in vivo—the authors demonstrate that hair cells manifest electrophysiological properties similar to hair cells in vivo and form synaptic junctions with neurons. The organoids form epithelia of the vestibular sensory kind by default. Type II hair cells and JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 3, SEPTEMBER 2017

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supporting cells are present, but type I hair cells are not. It is hoped that the inner-ear organoids will provide information about embryonic development and enable screening of therapeutic agents that may damage hair cells. Roper J, Tammela T, Cetinbas N M, Akkad A, Roghanian A, Rickelt S, Almeqdadi M, Wu K, Oberli M A, Sanchez-Rivera F, Park Y K, Liang X, Eng G, Taylor M S, Azimi R, Kedrin D, Neupane R, Beyaz S, Sicinska E T, Suarez Y, Yoo J, Chen L, Zukerberg L, Katajisto P, Deshpande V, Bass A J, Tsichlis P N, Lees J, Langer R, Hynes R O, Chen J, Bhutkar A, Jacks T, Yilmaz O H. In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis. Nature Biotechnology 35;2017:569–576. O’Rourke K P, Loizou E, Livshits G, Schatoff E M, Baslan T, Manchado E, Simon J, Romesser P B, Leach B, Han T, Pauli C, Beltran H, Rubin M A, Dow L E, Lowe S W. Transplantation of engineered organoids enables rapid generation of metastatic mouse models of colorectal cancer. Nature Biotechnology 35;2017:577–582. Colorectal cancer develops in steps that begin with the formation of a nonmalignant adenoma, progresses to formation of a primary carcinoma comprised of increasingly invasive cells, and ends with metastatic seeding. Progression through this sequence is propelled by mutational events. It has been difficult to develop convenient, genetically engineered mouse models of colorectal cancer that produce tumors localized to the sites of human disease and that show progression in a manner amenable to observation. Two groups here demonstrate that colon organoids can be efficiently transplanted orthotopically (i.e., to locations where colon cancer develops in human patients) into immunocompetent recipient mice and there, recapitulate the entire oncogenic pathway within an experimentally convenient time frame. Appropriate mutations are engineered by CRISPR-Cas9 editing. The system provides flexibility in testing varying combinations of mutations in the engrafted organoids and ascertaining the effects of genetic modification of the host to study tumor–stroma interactions. IMAGING

Wei L, Chen Z, Shi L, Long R, Anzalone A V, Zhang L, Hu F, Yuste R, Cornish V W, Min W. Supermultiplex vibrational imaging. Nature 544;2017:465–470. The search continues for ways to increase the number of different molecules that can be simultaneously imaged in JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 3, SEPTEMBER 2017

biologic specimens by increasing selectivity yet without diminishing rapidity, sensitively, and resolution. Spontaneous Raman microscopy detects molecular vibrational transitions. Emission signals (frequency shifts between incident and scattered light) are much narrower than emission signals in fluorescence spectra and therefore, in principle, are capable of high selectivity. However, Raman signals are extremely weak. Raman microscopy is capable of resolution as good as fluorescence microscopy, but image acquisition takes an exceedingly long time. In a variant of the Raman technique that provides better sensitivity, the incident light stimulates emission of coherent radiation at a frequency that is Stokes shifted as a function of the frequency of molecular vibrations. This emitted radiation drives molecular vibrations. Wei et al. devise a new method that uses this coherent Raman imaging process but additionally, uses fluorescent labels. The sample is illuminated with 2 lasers. One produces radiation that stimulates vibrational resonance. This elicits a very strong signal from the fluorophore at a higher frequency, which is referred to as an anti-Stokes component. The second laser produces light at a wavelength close to, but not identical with, the electronic resonance wavelength of the fluorophore—the effect of which is to minimize fluorescence background. Only a narrow range of illuminating light frequencies induces a signal, so many different labels can be successfully imaged simultaneously. Sensitivities of detection as low as 250 nM are demonstrated with a time constant of just 1 ms. The authors show that their technique works with commercially available labels and create a panel of labels with 24 resolvable colors suitable for use with their methodology. Widespread adoption of the technique is anticipated, as it combines sensitivity, resolution, labeling versatility, and biocompatibility. Valm A M, Cohen S, Legant W R, Melunis J, Hershberg U, Wait E, Cohen A R, Davidson M W, Betzig E, Lippincott-Schwartz J. Applying systemslevel spectral imaging and analysis to reveal the organelle interactome. Nature 546;2017:162–167. Valm et al. assemble a suite of methods for studying the spatial relationships between cell organelles and the ways these relationships vary with time. With the use of cells expressing organelle-specific proteins with distinguishable fluorescence signatures, they follow lysosomes, mitochondria, endoplasmic reticulum, peroxisomes, and the Golgi apparatus. They concomitantly use a dye to label lipid droplets. Confocal microscopy is performed by pointscanning illumination of cells with 3 lasers simultaneously, and emitted light is collected in an array detector. Emission from 133

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the different fluorophores is deconvoluted computationally. This permits the frequency and locality of 2-, 3-, 4-, and 5-way interactions among organelles to be mapped. Time-lapse images acquired every 5 s provide information about dynamic changes in these spatial interactions. The authors extend the methodology to acquire 3D images of organelles in live cells with high spatial and temporal resolution by developing a spectral imaging implementation of lattice light sheet microscopy. The results quantitatively describe a pattern of contacts among the 6 organelles. The pattern may be interpreted in terms of the metabolic interactions among the organelles. The methodology opens a pathway for an investigation of how organelle inter-relationships respond to drugs, pathogens, and metabolic stress and how organelle relationships vary during cell migration, division, and differentiation. DRUG DISCOVERY AND CHARACTERIZATION

Han K, Jeng E E, Hess G T, Morgens D W, Li A, Bassik M C. Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise

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genetic interactions. Nature Biotechnology 35;2017: 463–474. The acquisition of resistance to chemotherapeutic agents by cancer cells has stimulated much interest in the identification of combinations of drugs to improve therapeutic outcomes. Standard drug-screening methods are of limited value, given the very large numbers of drug combinations of interest. Therefore, the authors of this paper turn to the use of the CRISPR-Cas9 gene deletion to induce knockouts in pairwise combinations in a robust, scalable manner. They construct a library of 490,000 paired sgRNAs and a pipeline for paired-end sequencing and scoring. They use this system to screen rapidly 21,321 pairs of drug targets for synthetic lethal interactions in chronic myeloid leukemia cells. The results identify targets for synergistic killing, such as a strong synergy between two Bcl-2 family members—BCL2L1 and MCL1—which act in parallel to block apoptosis. The results also constitute the largest mammalian genetic interaction map to date. The map will increase the scope of systematic analysis of genetic interaction networks.

JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 3, SEPTEMBER 2017

Article Watch: September 2017.

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