Cold Spring Harbor Laboratory Press Research Articles

How to Develop a Rare Disease Treatment

How to Develop a Rare Disease Treatment

Managing Information From Preprints: The Scholarly Record and the Public Need for Information (Especially During a Pandemic)

MODERATOR: Patricia K Baskin Executive Editor, Neurology Journals American Academy of Neurology Minneapolis, Minnesota SPEAKERS: John R Inglis Cold Spring Harbor Laboratory Cold Spring Harbor, New York Bruce T Rosenblum Inera Somerville, Massachusetts Iratxe Puebla ASAPBio San Francisco, California REPORTER: Tony Alves Hopedale, Massachusetts The session entitled “Managing Information from Preprints: The Scholarly Record and the Public Need for Information (Especially During a Pandemic)” was held on May 3, 2021 at the Council of Science Editors Annual Meeting. Moderated by Patty Baskin, Executive Editor of the Neurology® Journals, the session featured presentations from John Inglis, Co-founder of bioRxiv and medRxiv, and Executive Director of Cold Spring Harbor Laboratory Press; Iratxe Puebla, Associate Director of ASAPbio; and Bruce Rosenblum, Vice President of Content and Workflow Solutions at Inera, an Atypon Company. During the COVID-19 pandemic, the usage and popularity of preprint servers has grown. Preprints have been common in disciplines such as physics, astronomy, and mathematics for decades, via the arXiv1 preprint server, but they are a somewhat new phenomenon in biology and medicine. Preprints allow for an early look at new research, often before authors submit their manuscripts to scholarly journals for evaluation and peer review. Because readers have access to research before it is peer reviewed, confusion and misconceptions around preprints may arise, particularly among the general public. John Inglis presented first and provided a comprehensive overview of how preprints are transforming research communications, using bioRxiv2 and medRxiv3 from Cold Spring Harbor Laboratory (CSHL) as examples. Inglis spoke of […]

Context-aware synthetic biology by controller design: Engineering the mammalian cell.

The rise of systems biology has ushered a new paradigm: the view of the cell as a system that processes environmental inputs to drive phenotypic outputs. Synthetic biology provides a complementary approach, allowing us to program cell behavior through the addition of synthetic genetic devices into the cellular processor. These devices, and the complex genetic circuits they compose, are engineered using a design-prototype-test cycle, allowing for predictable device performance to be achieved in a context-dependent manner. Within mammalian cells, context effects impact device performance at multiple scales, including the genetic, cellular, and extracellular levels. In order for synthetic genetic devices to achieve predictable behaviors, approaches to overcome context dependence are necessary. Here, we describe control systems approaches for achieving context-aware devices that are robust to context effects. We then consider cell fate programing as a case study to explore the potential impact of context-aware devices for regenerative medicine applications.

About the Master.

See related article on p. 130Doug Green is the Peter C. Doherty Endowed Chair of Immunology at St. Jude Children's Research Hospital (Memphis, TN). Prior to this, he was Head of the Division of Cellular Immunology at the La Jolla Institute for Allergy and Immunology. Doug received his Bsc (1977) and PhD (1981) from Yale University (New Haven, CT), followed by postdoctoral work in experimental surgery and marine ecology, also at Yale. He joined the faculty at the University of Alberta in Edmonton in 1985, where he attained the rank of Associate Professor. He moved to what is now the La Jolla Institute of Immunology in 1990 and to St. Jude in 2005. His research has focused on the process of active cell death and cell survival, extending from the role of cell death in the regulation of cancer and immune responses in the whole organism to the fundamental molecular events directing the death of the cell. More recently, he discovered the process of LC3-associated phagocytosis, which links the autophagy pathway to phagosome maturation. Other areas of intense interest include regulated necrosis and metabolic reprogramming during asymmetric division in T lymphocytes. He has published over 600 papers, chapters, commentaries, and books and is an ISI “highly cited” investigator. In 2017, he was awarded the Jürg Tschopp Prize for research on cell death. He is an honorary fellow of Trinity College, Dublin, an honorary Einstein Professor in China, and a foreign fellow of the Royal Society of Canada and received an honorary PhD from Tor Vergata University of Rome. His most recent book is “Apoptosis and Other Cell Death Mechanisms: Means to an End,” published in 2018 by Cold Spring Harbor Laboratory Press. His wife, Rona Mogil (PhD, Yale, 1985), and daughter, Maggie Green (BA, Yale, 2014), are much smarter than he is and display great patience in putting up with him.

Neutrophil myeloperoxidase harbors distinct site-specific peculiarities in its glycosylation

Anti-neutrophil cytoplasmic autoantibodies (ANCAs) are directed against lysosomal components of neutrophils. ANCAs directed to proteinase 3 and myeloperoxidase (MPO) in particular are associated with distinct forms of small vessel vasculitides. MPO is an abundant neutrophil-derived heme protein that is part of the antimicrobial defense system. The protein is typically present in the azurophilic granules of neutrophils, but a large portion may also enter the extracellular space. It remains unclear why MPO is frequently the target of antibody-mediated autoimmune responses. MPO is a homodimeric glycoprotein, posttranslationally modified with complex sugars at specific sites. Glycosylation can strongly influence protein function, affecting its folding, receptor interaction, and backbone accessibility. MPO potentially can be heavily modified as it harbors 5 putative N-glycosylation sites (10 in the mature dimer). Although considered important for MPO structure and function, the full scope and relative abundance of the glycans attached to MPO is unknown. Here, combining bottom-up glycoproteomics and native MS approaches, we structurally characterized MPO from neutrophils of healthy human donors. We quantified the relative occupancy levels of the glycans at each of the five sites and observed complex heterogeneity and site-specific glycosylation. In particular, we detected glycosylation phenotypes uncommon for glycoproteins in the extracellular space, such as a high abundance of phosphorylated high-mannose species and severely truncated small glycans having the size of paucimannose or smaller. We hypothesize that the atypical glycosylation pattern found on MPO might contribute to its specific processing and presentation as a self-antigen by antigen-presenting cells.

Preprints in Context

Watch the VIDEO.The big story in scholarly publishing of the last years was the preprint revolution. Researchers demanded a faster channel for the early communication of their results, journal editors revised their policies to accept manuscripts also published as preprints, there was an explosion of new platforms and infrastructure, and Crossref gave the old/new species of not-really published articles a DOI. Funders even jumped into the fray, allowing researchers to use their preprints in grant proposals. The non-profit Center of Open Science launched a platform for new preprint servers, and publishers are jumping into the arena with preprint repositories like Cold Spring Harbor Laboratory Press’s bioRxiv, PeerJ Preprints, ACS’s ChemRxiv, MDPI’s Preprint.org and Elsevier’s acquisition of SSRN. These developments create new challenges for content aggregators and discovery platforms, librarians, publishers, and readers, including persistent identification of the version of record, aggregation of citations and metrics across article versions, and questions around copyright. The wide proliferation of preprint repositories across different sources also makes it difficult for researchers to reach their peers with their early results. As an aggregator and interactive discovery platform, ScienceOpen has been working to place preprints in the context of the published literature with quick preprint-filters on keyword searches and first efforts to aggregate metrics. We further provide a framework to peer review preprints in a portable system with a Crossref DOI for each peer review report, as well as editorial management of preprints in thematic collections. By showing how preprints fit in to the academic record in a broad discovery database, we are able to bring them to the attention of those searching for the newest research and more clearly highlight their status as open for peer review. Increased visibility through contextualization is our value proposition for publishers, authors and readers of preprints in 2018. We explore the disruptive potential of preprints from our experience with ScienceOpen’s discovery platform.

Automated Glycan Assembly of Oligo-N-Acetyllactosamine and Keratan Sulfate Probes to Study Virus-Glycan Interactions

Summary Oligo- N -acetyllactosamine (LacNAc) and keratan sulfate (KS) glycans exert crucial functions in disease-relevant processes, including cancer formation, inflammation, and viral infection. To facilitate structure-activity studies with these glycans, we established a universal strategy to synthesize linear and branched LacNAc as well as differentially sulfated KS oligosaccharides by automated glycan assembly. We synthesized oligosaccharides as long as hexamers by combining four monosaccharide building blocks. Key to the strategy was installing three orthogonal protection groups, 9-fluorenylmethoxycarbonyl (Fmoc), levulinoyl (Lev) ester, and 2-naphthylmethyl (Nap) ether, which were selectively removed from a common oligosaccharide precursor for differential sulfation. Microarrays presenting the synthetic oligosaccharides revealed a specific interaction between a disulfated KS tetrasaccharide and the adeno-associated virus AAVrh10 gene-therapy vector, which was further corroborated by surface plasmon resonance studies. Thus, KS represents a novel receptor candidate for AAVrh10. These insights could have implications for cell-type-specific gene-delivery approaches.

Book review: Epigenetics (second edition, eds. Allis, Caparros, Jenuwein, Reinberg)

Book review: Epigenetics (second edition, eds. Allis, Caparros, Jenuwein, Reinberg)

How to Become a Control FreakReview and commentary on Signal Transduction: Principles, Pathways, and Processes , edited by Lewis C. Cantley, Tony Hunter, Richard Sever,and Jeremy Thorner. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2014. 452 pp. ISBN: 978-0-879699-01-7

A new textbook offers up-to-date, comprehensive, but unfinished, wiring diagrams for budding cellular electricians.

Inhibition of the Ribonuclease H Activity of HIV-1 Reverse Transcriptase by GSK5750 Correlates with Slow Enzyme-Inhibitor Dissociation

Compounds that efficiently inhibit the ribonuclease (RNase) H activity of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have yet to be developed. Here, we demonstrate that GSK5750, a 1-hydroxy-pyridopyrimidinone analog, binds to the enzyme with an equilibrium dissociation constant (K(d)) of ~400 nM. Inhibition of HIV-1 RNase H is specific, as DNA synthesis is not affected. Moreover, GSK5750 does not inhibit the activity of Escherichia coli RNase H. Order-of-addition experiments show that GSK5750 binds to the free enzyme in an Mg(2+)-dependent fashion. However, as reported for other active site inhibitors, binding of GSK5750 to a preformed enzyme-substrate complex is severely compromised. The bound nucleic acid prevents access to the RNase H active site, which represents a possible biochemical hurdle in the development of potent RNase H inhibitors. Previous studies suggested that formation of a complex with the prototypic RNase H inhibitor β-thujaplicinol is slow, and, once formed, it dissociates rapidly. This unfavorable kinetic behavior can limit the potency of RNase H active site inhibitors. Although the association kinetics of GSK5750 remains slow, our data show that this compound forms a long lasting complex with HIV-1 RT. We conclude that slow dissociation of the inhibitor and HIV-1 RT improves RNase H active site inhibitors and may circumvent the obstacle posed by the inability of these compounds to bind to a preformed enzyme-substrate complex.

A Rhomboid Protease Gene Deletion Affects a Novel Oligosaccharide N-Linked to the S-layer Glycoprotein of Haloferax volcanii

Rhomboid proteases occur in all domains of life; however, their physiological role is not completely understood, and nothing is known of the biology of these enzymes in Archaea. One of the two rhomboid homologs of Haloferax volcanii (RhoII) is fused to a zinc finger domain. Chromosomal deletion of rhoII was successful, indicating that this gene is not essential for this organism; however, the mutant strain (MIG1) showed reduced motility and increased sensitivity to novobiocin. Membrane preparations of MIG1 were enriched in two glycoproteins, identified as the S-layer glycoprotein and an ABC transporter component. The H. volcanii S-layer glycoprotein has been extensively used as a model to study haloarchaeal protein N-glycosylation. HPLC analysis of oligosaccharides released from the S-layer glycoprotein after PNGase treatment revealed that MIG1 was enriched in species with lower retention times than those derived from the parent strain. Mass spectrometry analysis showed that the wild type glycoprotein released a novel oligosaccharide species corresponding to GlcNAc-GlcNAc(Hex)2-(SQ-Hex)6 in contrast to the mutant protein, which contained the shorter form GlcNAc2(Hex)2-SQ-Hex-SQ. A glycoproteomics approach of the wild type glycopeptide fraction revealed Asn-732 peptide fragments linked to the sulfoquinovose-containing oligosaccharide. This work describes a novel N-linked oligosaccharide containing a repeating SQ-Hex unit bound to Asn-732 of the H. volcanii S-layer glycoprotein, a position that had not been reported as glycosylated. Furthermore, this study provides the first insight on the biological role of rhomboid proteases in Archaea, suggesting a link between protein glycosylation and this protease family.

Identification of Ligand Specificity Determinants in Lantibiotic Bovicin HJ50 and the Receptor BovK, a Multitransmembrane Histidine Kinase

Lantibiotic bovicin HJ50 is produced by Streptococcus bovis HJ50 and acts as the extracellular signal to autoregulate its own biosynthesis through BovK/R two-component system. Bovicin HJ50 shows a linear N-terminal and glubolar C-terminal structure, and the sensor histidine kinase BovK contains eight transmembrane segments lacking any extensive surface-exposed sensory domain. The signal recognition mechanism between bovicin HJ50 and BovK is still unknown. We performed saturated alanine scanning mutagenesis and other amino acid substitutions on bovicin HJ50 using a semi-in vitro biosynthesis. Results of the mutants inducing activities indicated that several charged and hydrophobic amino acids in ring B of bovicin HJ50, as well as two glycines were key residues to recognize BovK. Circular dichroism analyses indicated that both glycines contributed to bovicin HJ50 structural changes in the membrane. Biotin-labeled bovicin HJ50 could interact with the N-terminal sensor of BovK, and several charged residues and a conserved hydrophobic region in the N-terminal portion of BovK sensor domain were important for interacting with the signal bovicin HJ50. By combining the results, we suggested a mechanism of bovicin HJ50 recognizing and activating BovK mainly through electrostatic and hydrophobic interactions.

Wishful Thinking, The Will to Power, and Skepticism in the History of Human Genetics in the USSRA review of The Dawn of Human Genetics. By V. V. Babkov; translated by, Victor Fet; edited by, James Schwartz. Cold Spring Harbor (New York): Cold Spring Harbor Laboratory Press. $69.00. xxxii + 775 p. + 16 pl.; ill.; index. ISBN: 978-1-936113-70-5. [Originally published

Previous articleNext article No AccessNew Biological BooksWishful Thinking, The Will to Power, and Skepticism in the History of Human Genetics in the USSR A review of The Dawn of Human Genetics. By V. V. Babkov; translated by, Victor Fet; edited by, James Schwartz. Cold Spring Harbor (New York): Cold Spring Harbor Laboratory Press. $69.00. xxxii + 775 p. + 16 pl.; ill.; index. ISBN: 978-1-936113-70-5. [Originally published in Russia in 2005.] 2013.Elof Axel CarlsonElof Axel CarlsonBiochemistry & Cell Biology, Stony Brook University, Stony Brook, New York 11794 and Institute for Advanced Study, Indiana University, Bloomington, Indiana 47406 USA [email protected] Search for more articles by this author PDFPDF PLUSFull Text Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Quarterly Review of Biology Volume 88, Number 4December 2013 Published in association with Stony Brook University Article DOIhttps://doi.org/10.1086/673759 Views: 16Total views on this site Copyright © 2013 by The University of Chicago Press. All rights reserved.PDF download Crossref reports no articles citing this article.

Polyamines Are Critical for the Induction of the Glutamate Decarboxylase-dependent Acid Resistance System in Escherichia coli

As part of our studies on the biological functions of polyamines, we have used a mutant of Escherichia coli that lacks all the genes for polyamine biosynthesis for a global transcriptional analysis on the effect of added polyamines. The most striking early response to the polyamine addition is the increased expression of the genes for the glutamate-dependent acid resistance system (GDAR) that is important for the survival of the bacteria when passing through the acid environment of the stomach. Not only were the two genes for glutamate decarboxylases (gadA and gadB) and the gene for glutamate-γ-aminobutyrate antiporter (gadC) induced by the polyamine addition, but the various genes involved in the regulation of this system were also induced. We confirmed the importance of polyamines for the induction of the GDAR system by direct measurement of glutamate decarboxylase activity and acid survival. The effect of deletions of the regulatory genes on the GDAR system and the effects of overproduction of two of these genes were also studied. Strikingly, overproduction of the alternative σ factor rpoS and of the regulatory gene gadE resulted in very high levels of glutamate decarboxylase and almost complete protection against acid stress even in the absence of any polyamines. Thus, these data show that a major function of polyamines in E. coli is protection against acid stress by increasing the synthesis of glutamate decarboxylase, presumably by increasing the levels of the rpoS and gadE regulators.

Inactivation of the Bacterial RNA Polymerase Due to Acquisition of Secondary Structure by the ω Subunit

The widely conserved ω subunit encoded by rpoZ is the smallest subunit of Escherichia coli RNA polymerase (RNAP) but is dispensable for bacterial growth. Function of ω is known to be substituted by GroEL in ω-null strain, which thus does not exhibit a discernable phenotype. In this work, we report isolation of ω variants whose expression in vivo leads to a dominant lethal phenotype. Studies show that in contrast to ω, which is largely unstructured, ω mutants display substantial acquisition of secondary structure. By detailed study with one of the mutants, ω6 bearing N60D substitution, the mechanism of lethality has been deciphered. Biochemical analysis reveals that ω6 binds to β' subunit in vitro with greater affinity than that of ω. The reconstituted RNAP holoenzyme in the presence of ω6 in vitro is defective in transcription initiation. Formation of a faulty RNAP in the presence of mutant ω results in death of the cell. Furthermore, lethality of ω6 is relieved in cells expressing the rpoC2112 allele encoding β'2112, a variant β' bearing Y457S substitution, immediately adjacent to the β' catalytic center. Our results suggest that the enhanced ω6-β' interaction may perturb the plasticity of the RNAP active center, implicating a role for ω and its flexible state.

Mutation of Light-dependent Phosphorylation Sites of the Drosophila Transient Receptor Potential-like (TRPL) Ion Channel Affects Its Subcellular Localization and Stability

The Drosophila phototransduction cascade terminates in the opening of the ion channel transient receptor potential (TRP) and TRP-like (TRPL). Contrary to TRP, TRPL undergoes light-dependent subcellular trafficking between rhabdomeric photoreceptor membranes and an intracellular storage compartment, resulting in long term light adaptation. Here, we identified in vivo phosphorylation sites of TRPL that affect TRPL stability and localization. Quantitative mass spectrometry revealed a light-dependent change in the TRPL phosphorylation pattern. Mutation of eight C-terminal phosphorylation sites neither affected multimerization of the channels nor the electrophysiological response of flies expressing the mutated channels. However, these mutations resulted in mislocalization and enhanced degradation of TRPL after prolonged dark-adaptation. Mutation of subsets of the eight C-terminal phosphorylation sites also led to a reduction of TRPL content and partial mislocalization in the dark. This suggests that a light-dependent switch in the phosphorylation pattern of the TRPL channel mediates stable expression of TRPL in the rhabdomeres upon prolonged dark-adaptation.

Carnal Light

Carnal Light

Yeast Eukaryotic Initiation Factor 4B (eIF4B) Enhances Complex Assembly between eIF4A and eIF4G in Vivo

Translation initiation factor eIF4F (eukaryotic initiation factor 4F), composed of eIF4E, eIF4G, and eIF4A, binds to the m(7)G cap structure of mRNA and stimulates recruitment of the 43S preinitiation complex and subsequent scanning to the initiation codon. The HEAT domain of eIF4G stabilizes the active conformation of eIF4A required for its RNA helicase activity. Mammalian eIF4B also stimulates eIF4A activity, but this function appears to be lacking in yeast, making it unclear how yeast eIF4B (yeIF4B/Tif3) stimulates translation. We identified Ts(-) mutations in the HEAT domains of yeast eIF4G1 and eIF4G2 that are suppressed by overexpressing either yeIF4B or eIF4A, whereas others are suppressed only by eIF4A overexpression. Importantly, suppression of HEAT domain substitutions by yeIF4B overexpression was correlated with the restoration of native eIF4A·eIF4G complexes in vivo, and the rescue of specific mutant eIF4A·eIF4G complexes by yeIF4B was reconstituted in vitro. Association of eIF4A with WT eIF4G in vivo also was enhanced by yeIF4B overexpression and was impaired in cells lacking yeIF4B. Furthermore, we detected native complexes containing eIF4G and yeIF4B but lacking eIF4A. These and other findings lead us to propose that yeIF4B acts in vivo to promote eIF4F assembly by enhancing a conformation of the HEAT domain of yeast eIF4G conducive for stable binding to eIF4A.

Identification of Drosophila and Human 7-Methyl GMP-specific Nucleotidases

Turnover of mRNA releases, in addition to the four regular nucleoside monophosphates, the methylated cap nucleotide in the form of 7-methylguanosine monophosphate (m(7)GMP) or diphosphate (m(7)GDP). The existence of pathways to eliminate the modified nucleotide seems likely, as its incorporation into nucleic acids is undesirable. Here we describe a novel 5' nucleotidase from Drosophila that cleaves m(7)GMP to 7-methylguanosine and inorganic phosphate. The enzyme, encoded by the predicted gene CG3362, also efficiently dephosphorylates CMP, although with lower apparent affinity; UMP and the purine nucleotides are poor substrates. The enzyme is inhibited by elevated concentrations of AMP and also cleaves m(7)GDP to the nucleoside and two inorganic phosphates, albeit less efficiently. CG3362 has equivalent sequence similarity to two human enzymes, cytosolic nucleotidase III (cNIII) and the previously uncharacterized cytosolic nucleotidase III-like (cNIII-like). We show that cNIII-like also displays 5' nucleotidase activity with a high affinity for m(7)GMP. CMP is a slightly better substrate but again with a higher K(m). The activity of cNIII-like is stimulated by phosphate. In contrast to cNIII-like, cNIII and human cytosolic nucleotidase II do not accept m(7)GMP as a substrate. We suggest that the m(7)G-specific nucleotidases protect cells against undesired salvage of m(7)GMP and its incorporation into nucleic acids.

MicroRNA‐independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA‐binding protein

Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). Here, we describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA-binding protein. We show that Smaug interacts with the Ago1 protein, and that Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, our data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA-independent manner, thereby repressing translation.