Journal of Molecular Biology

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  • Gradual folding of an off-pathway molten globule detected at the single-molecule level
    [Jul 2015]

    Publication date: Available online 8 July 2015
    Source:Journal of Molecular Biology

    Author(s): Simon Lindhoud, Menahem Pirchi, Adrie H. Westphal, Gilad Haran, Carlo P.M. van Mierlo

    Molten globules (MGs) are compact, partially folded intermediates that are transiently present during folding of many proteins. These intermediates reside on or off the folding pathway to native protein. Conformational evolution during folding of off-pathway MGs is largely unexplored. Here, we characterize the denaturant-dependent structure of apoflavodoxin’s off-pathway MG. Using single-molecule FRET we follow conversion of unfolded species into MG down to denaturant concentrations that favor formation of native protein. Under strongly denaturing conditions, FRET histograms show a single peak, arising from unfolded protein. The smFRET efficiency distribution shifts to higher value upon decreasing denaturant concentration, because the MG folds. Strikingly, upon approaching native conditions the FRET efficiency of the MG rises above that of native protein. Thus, smFRET exposes the misfolded nature of apoflavodoxin’s off-pathway MG. We show that conversion from unfolded to MG protein is a gradual, second-order like process that simultaneously involves separate regions within the polypeptide.
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    Categories: Journal Articles
  • A New Versatile Immobilization Tag Based on the Ultra High Affinity and Reversibility of the Calmodulin–Calmodulin Binding Peptide Interaction
    [Jul 2015]

    Publication date: Available online 6 July 2015
    Source:Journal of Molecular Biology

    Author(s): Somnath Mukherjee, Marcin Ura, Robert J. Hoey, Anthony A. Kossiakoff

    Reversible, high-affinity immobilization tags are critical tools for myriad biological applications. However, inherent issues are associated with a number of the current methods of immobilization. Particularly, a critical element in phage display sorting is functional immobilization of target proteins. To circumvent these problems, we have used a mutant (N5A) of calmodulin binding peptide (CBP) as an immobilization tag in phage display sorting. The immobilization relies on the ultra high affinity of calmodulin to N5A mutant CBP (RWKKNFIAVSAANRFKKIS) in presence of calcium (K D ~2pM), which can be reversed by EDTA allowing controlled “capture and release” of the specific binders. To evaluate the capabilities of this system, we chose eight targets, some of which were difficult to overexpress and purify with other tags and some had failed in sorting experiments. In all cases, specific binders were generated using a Fab phage display library with CBP-fused constructs. K D values of the Fabs were in subnanomolar to low nanomolar (nM) ranges and were successfully used to selectively recognize antigens in cell-based experiments. Some of these targets were problematic even without any tag; thus, the fact that all led to successful selection endpoints means that borderline cases can be worked on with a high probability of a positive outcome. Taken together with examples of successful case specific, high-level applications like generation of conformation-, epitope- and domain-specific Fabs, we feel that the CBP tag embodies all the attributes of covalent immobilization tags but does not suffer from some of their well-documented drawbacks.
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  • Remaining mysteries of Molecular Biology: The role of polyamine metabolites in the cell
    [Jul 2015]

    Publication date: Available online 5 July 2015
    Source:Journal of Molecular Biology

    Author(s): Leonor Miller-Fleming, Viridiana Olin-Sandoval, Kate Campbell, Markus Ralser

    The polyamines (PAs) spermidine, spermine, putrescine and cadaverine, are an essential class of metabolites found throughout all kingdoms of life. In this comprehensive review we discuss their metabolism, their various intracellular functions, as well as their unusual and conserved regulatory features. These include regulation of translation via upstream open reading frames, the over-reading of stop-codons via ribosomal frameshifting, the existence of an antizyme and an antizyme inhibitor, ubiquitin-independent proteasomal degradation, metabolite-induced proteolysis, a complex bi-directional membrane transport system and a unique posttranslational modification - hypusination - that is believed to occur on a single protein only eIF-5A. Many of these features are broadly conserved indicating PA metabolism is both concentration critical and evolutionary ancient. When PA metabolism is disrupted, a plethora of cellular processes are affected, including transcription, translation, gene expression regulation, autophagy and stress resistance. As a result, the role of PAs has been associated with cell growth, ageing, memory performance, neurodegenerative diseases, metabolic disorders and cancer. Despite comprehensive studies addressing PA’s, a unifying concept to interpret their molecular role is however missing. The precise biochemical function of polyamines is thus one of the remaining mysteries of molecular cell biology.
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  • When too much ATP is bad for protein synthesis
    [Jul 2015]

    Publication date: Available online 4 July 2015
    Source:Journal of Molecular Biology

    Author(s): Mauricio H. Pontes, Anastasia Sevostyanova, Eduardo A. Groisman

    Adenosine triphosphate (ATP) is the energy currency of living cells. Even though ATP powers virtually all energy-dependent activity, most cellular ATP is utilized in protein synthesis via tRNA aminoacylation and GTP regeneration. Magnesium (Mg2+), the most common divalent cation in living cells, plays crucial roles in protein synthesis by maintaining the structure of ribosomes, participating in the biochemistry of translation initiation and functioning as a counter-ion for ATP. A non-physiological increase in ATP levels hinders growth in cells experiencing Mg2+ limitation because ATP is the most abundant nucleotide triphosphate in the cell and Mg2+ is also required for the stabilization of the cytoplasmic membrane and as a cofactor for essential enzymes. We propose that organisms cope with Mg2+ limitation by decreasing ATP levels and ribosome production, thereby reallocating Mg2+ to indispensable cellular processes.
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  • Charge Detection Mass Spectrometry Identifies Preferred Non-Icosahedral Polymorphs in the Self-Assembly of Woodchuck Hepatitis Virus Capsids
    [Jul 2015]

    Publication date: Available online 4 July 2015
    Source:Journal of Molecular Biology

    Author(s): Elizabeth E. Pierson, David Z. Keifer, Alexander A. Kukreja, Joseph C.-Y. Wang, Adam Zlotnick, Martin F. Jarrold

    Woodchuck hepatitis virus (WHV) is prone to aberrant assembly in vitro and can form a broad distribution of oversized particles. Characterizing aberrant assembly products is challenging because they are both large and heterogeneous. In this work, charge detection mass spectrometry (CDMS) is used to measure the distribution of WHV assembly products. CDMS is a single-particle technique where the masses of individual ions are determined from simultaneous measurement of each ion's charge and m/z (mass-to-charge) ratio. Under relatively aggressive, assembly promoting conditions, roughly half of the WHV assembly products are T =4 capsids composed of exactly 120 dimers while the other half are a broad distribution of larger species that extends to beyond 210 dimers. There are prominent peaks at around 132 dimers and at 150 dimers. In part, the 150 dimer complex can be attributed to elongating a T =4 capsid along its 5-fold axis by adding a ring of hexamers. However, most of the other features cannot be explained by existing models for hexameric defects. Cryo-electron microscopy provides evidence of elongated capsids. However, image analysis reveals that many of them are not closed but have “spiral-like” morphologies. The CDMS data indicate that oversized capsids have a preference for growth by addition of 3 or 4 dimers, probably by completion of hexameric vertices.
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    Categories: Journal Articles
  • Editorial Board
    [Jul 2015]

    Publication date: 3 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 13









    Categories: Journal Articles
  • Contents List
    [Jul 2015]

    Publication date: 3 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 13









    Categories: Journal Articles
  • The Tail-End Is Only the Beginning: NMR Study Reveals a Membrane-Bound State of BCL-XL
    [Jul 2015]

    Publication date: 3 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 13

    Author(s): R. Blake Hill, Kevin R. MacKenzie, Megan Cleland Harwig







    Categories: Journal Articles
  • Conformation of BCL-XL upon Membrane Integration
    [Jul 2015]

    Publication date: 3 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 13

    Author(s): Yong Yao, Lynn M. Fujimoto, Nathan Hirshman, Andrey A. Bobkov, Antonella Antignani, Richard J. Youle, Francesca M. Marassi

    BCL-XL is an anti-apoptotic BCL-2 family protein found both in the cytosol and bound to intracellular membranes. Structural studies of BCL-XL have advanced by deleting its hydrophobic C-terminus and adding detergents to enhance solubility. However, since the C-terminus is essential for function and detergents strongly affect structure and activity, the molecular mechanisms controlling intracellular localization and cytoprotective activity are incompletely understood. Here we describe the conformations and ligand binding activities of water-soluble and membrane-bound BCL-XL, with its complete C-terminus, in detergent-free environments. We show that the C-terminus interacts with a conserved surface groove in the water-soluble state of the protein and inserts across the phospholipid bilayer in the membrane-bound state. Contrary to current models, membrane binding does not induce a conformational change in the soluble domain and both states bind a known ligand with affinities that are modulated by the specific state of the protein.
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    Categories: Journal Articles
  • Genetic Basis of Common Human Disease: Insight into the Role of Missense SNPs from Genome-Wide Association Studies
    [Jul 2015]

    Publication date: 3 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 13

    Author(s): Lipika R. Pal, John Moult

    Recent genome-wide association studies (GWAS) have led to the reliable identification of single nucleotide polymorphisms (SNPs) at a number of loci associated with increased risk of specific common human diseases. Each such locus implicates multiple possible candidate SNPs for involvement in disease mechanism. A variety of mechanisms may link the presence of an SNP to altered in vivo gene product function and hence contribute to disease risk. Here, we report an analysis of the role of one of these mechanisms, missense SNPs (msSNPs) in proteins in seven complex trait diseases. Linkage disequilibrium information was used to identify possible candidate msSNPs associated with increased disease risk at each of 356 loci for the seven diseases. Two computational methods were used to estimate which of these SNPs has a significant impact on in vivo protein function. 69% of the loci have at least one candidate msSNP and 33% have at least one predicted high-impact msSNP. In some cases, these SNPs are in well-established disease-related proteins, such as MST1 (macrophage stimulating 1) for Crohn's disease. In others, they are in proteins identified by GWAS as likely candidates for disease relevance, but previously without known mechanism, such as ADAMTS13 (ADAM metallopeptidase with thrombospondin type 1 motif, 13) for coronary artery disease. In still other cases, the missense SNPs are in proteins not previously suggested as disease candidates, such as TUBB1 (tubulin, beta 1, class VI) for hypertension. Together, these data support a substantial role for this class of SNPs in susceptibility to common human disease.
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  • RING E3-Catalyzed E2 Self-Ubiquitination Attenuates the Activity of Ube2E Ubiquitin-Conjugating Enzymes
    [Jul 2015]

    Publication date: 3 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 13

    Author(s): Prerana Agarwal Banka, Adaitya Prasad Behera, Sayani Sarkar, Ajit B. Datta

    Ubiquitination of a target protein is accomplished through sequential actions of the E1, E2s, and the E3s. E2s dictate the modification topology while E3 ligases confer substrate specificity and recruit the cognate E2. Human genome codes for ~35 different E2 proteins; all of which contain the characteristic ubiquitin-conjugating UBC core domain sufficient for catalysis. Many of these E2 enzymes also have N- or C-terminal extensions; roles of which are not very well understood. We show that the N-terminal extension of Ube2E1 undergoes intramolecular auto-ubiquitination. This self-ubiquitination activity is enhanced in the presence of interacting RING E3 ligases and results in a progressive attenuation of the E2 activity toward substrate/E3 modification. We also find that the N-terminal ubiquitination sites are conserved in all the three Ube2Es and replacing them with arginine renders all three full-length Ube2Es equally active as their core UBC domains. Based on these results, we propose that E3-catalyzed self-ubiquitination acts as a key regulatory mechanism that controls the activity of Ube2E class of ubiquitin E2s.
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  • E. coli Gyrase Fails to Negatively Supercoil Diaminopurine-Substituted DNA
    [Jul 2015]

    Publication date: 3 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 13

    Author(s): Mónica Fernández-Sierra, Qing Shao, Chandler Fountain, Laura Finzi, David Dunlap

    Type II topoisomerases modify DNA supercoiling, and crystal structures suggest that they sharply bend DNA in the process. Bacterial gyrases are a class of type II topoisomerases that can introduce negative supercoiling by creating a wrap of DNA before strand passage. Isoforms of these essential enzymes were compared to reveal whether they can bend or wrap artificially stiffened DNA. Escherichia coli gyrase and human topoisomerase IIα were challenged with normal DNA or stiffer DNA produced by polymerase chain reaction reactions in which diaminopurine (DAP) replaced adenine deoxyribonucleotide triphosphates. On single DNA molecules twisted with magnetic tweezers to create plectonemes, the rates or pauses during relaxation of positive supercoils in DAP-substituted versus normal DNA were distinct for both enzymes. Gyrase struggled to bend or perhaps open a gap in DAP-substituted DNA, and segments of wider DAP DNA may have fit poorly into the N-gate of the human topoisomerase IIα. Pauses during processive activity on both types of DNA exhibited ATP dependence consistent with two pathways leading to the strand-passage-competent state with a bent gate segment and a transfer segment trapped by an ATP-loaded and latched N-gate. However, E. coli DNA gyrase essentially failed to negatively supercoil 35% stiffer DAP DNA.
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  • Antiparallel β-Sheet Structure within the C-Terminal Region of 42-Residue Alzheimer's Amyloid-β Peptides When They Form 150-kDa Oligomers
    [Jul 2015]

    Publication date: 3 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 13

    Author(s): Danting Huang, Maxwell I. Zimmerman, Patricia K. Martin, A.Jeremy Nix, Terrone L. Rosenberry, Anant K. Paravastu

    Understanding the molecular structures of amyloid-β (Aβ) oligomers and underlying assembly pathways will advance our understanding of Alzheimer's disease (AD) at the molecular level. This understanding could contribute to disease prevention, diagnosis, and treatment strategies, as oligomers play a central role in AD pathology. We have recently presented a procedure for production of 150-kDa oligomeric samples of Aβ(1-42) (the 42-residue variant of the Aβ peptide) that are compatible with solid-state nuclear magnetic resonance (NMR) analysis, and we have shown that these oligomers and amyloid fibrils differ in intermolecular arrangement of β-strands. Here we report new solid-state NMR constraints that indicate antiparallel intermolecular alignment of β-strands within the oligomers. Specifically, 150-kDa Aβ(1-42) oligomers with uniform 13C and 15N isotopic labels at I32, M35, G37, and V40 exhibit β-strand secondary chemical shifts in 2-dimensional (2D) finite-pulse radiofrequency-driven recoupling NMR spectra, spatial proximities between I32 and V40 as well as between M35 and G37 in 2D dipolar-assisted rotational resonance spectra, and close proximity between M35 Hα and G37 Hα in 2D CHHC spectra. Furthermore, 2D dipolar-assisted rotational resonance analysis of an oligomer sample prepared with 30% labeled peptide indicates that the I32-V40 and M35-G37 contacts are between residues on different molecules. We employ molecular modeling to compare the newly derived experimental constraints with previously proposed geometries for arrangement of Aβ molecules into oligomers.
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  • Catalytic role of the substrate defines specificity of therapeutic L-asparaginase (L-ASP)
    [Jul 2015]

    Publication date: Available online 2 July 2015
    Source:Journal of Molecular Biology

    Author(s): Andriy Anishkin, Juan M. Vanegas, David M. Rogers, Philip L. Lorenzi, Wai Kin Chan, Preeti Purwaha, John N. Weinstein, Sergei Sukharev, Susan B. Rempe

    Type II bacterial L-asparaginases (L-ASP) have played an important therapeutic role in cancer treatment for over four decades, yet their exact reaction mechanism remains elusive. L-ASP from E. coli deamidates asparagine (Asn) and glutamine, with a ~104 higher specificity (k cat/K m) for asparagine despite only one methylene difference in length. Through a sensitive kinetic approach, we quantify competition among the substrates and interpret its clinical role. To understand specificity, we use molecular simulations characterize enzyme interactions with substrates and a product (aspartate). We present evidence that the aspartate product in the crystal structure of L-ASP exists in an unusual α-COOH protonation state. Consequently, the set of enzyme-product interactions found in the crystal structure, which guided prior mechanistic interpretations, differs from those observed in dynamic simulations of the enzyme with the substrates. Finally, we probe the initial nucleophilic attack with ab initio simulations. The unusual protonation state reappears, suggesting that crystal structures (wild-type and a T89V mutant) represent intermediate steps rather than initial binding. Also, a proton transfers spontaneously to Asn, advancing a new hypothesis that the substrate’s α-carboxyl serves as a proton acceptor and activates one of the catalytic threonines during L-ASP’s nucleophilic attack on the amide carbon. That hypothesis explains for the first time why proximity of the substrate α-COO- group to the carboxamide is absolutely required for catalysis. The substrate’s catalytic role is likely the determining factor in enzyme specificity as it constrains the allowed distance between the backbone carboxyl and the amide carbon of any L-ASP substrate.
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    Categories: Journal Articles
  • One-Dimensional Sliding of p53 Along DNA Is Accelerated in the Presence of Ca2+ or Mg2+ at Millimolar Concentrations
    [Jul 2015]

    Publication date: Available online 2 July 2015
    Source:Journal of Molecular Biology

    Author(s): Agato Murata, Yuji Ito, Risa Kashima, Saori Kanbayashi, Kei Nanatani, Chihiro Igarashi, Masaki Okumura, Kenji Inaba, Takashi Tokino, Satoshi Takahashi, Kiyoto Kamagata

    One-dimensional (1D) sliding of the tumor suppressor p53 along DNA is an essential dynamics required for its efficient search for the binding sites in the genome. To address how the search process of p53 is affected by the changes in the concentration of Mg2+ and Ca2+ after the cell damages, we investigated its sliding dynamics at different concentrations of the divalent cations. The 1D sliding trajectories of p53 along the stretched DNA were measured by using single-molecule fluorescence microscopy. The averaged diffusion coefficient calculated from the mean square displacement of p53 on DNA increased significantly at the higher concentration of Mg2+ or Ca2+, indicating that the divalent cations accelerate the sliding likely by weakening the DNA–p53 interaction. In addition, two distributions were identified in the displacement of the observed trajectories of p53, demonstrating the presence of the fast and slow sliding modes having large and small diffusion coefficients, respectively. A coreless mutant of p53, in which the core domain was deleted, showed only a single mode whose diffusion coefficient is about twice that of the fast mode for the full-length p53. Thus, the two modes are likely the result of the tight and loose interactions between the core domain of p53 and DNA. These results demonstrated clearly that the 1D sliding dynamics of p53 is strongly dependent on the concentration of Mg2+ and Ca2+, which maintains the search distance of p53 along DNA in cells that lost homeostatic control of the divalent cations.
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    Categories: Journal Articles
  • Structure of Hepatitis C Virus Envelope Glycoprotein E1 Antigenic Site 314–324 in Complex with Antibody IGH526
    [Jul 2015]

    Publication date: Available online 30 June 2015
    Source:Journal of Molecular Biology

    Author(s): Leopold Kong, Rameshwar U. Kadam, Erick Giang, Tinashe B. Ruwona, Travis Nieusma, Jeffrey C. Culhane, Robyn L. Stanfield, Philip E. Dawson, Ian A. Wilson, Mansun Law

    Hepatitis C virus (HCV) is a positive-strand RNA virus within the Flaviviridae family. The viral “spike” of HCV is formed by two envelope glycoproteins, E1 and E2, which together mediate viral entry by engaging host receptors and undergoing conformational changes to facilitate membrane fusion. While E2 can be readily produced in the absence of E1, E1 cannot be expressed without E2 and few reagents, including monoclonal antibodies (mAbs), are available for study of this essential HCV glycoprotein. A human mAb to E1, IGH526, was previously reported to cross-neutralize different HCV isolates, and therefore, we sought to further characterize the IGH526 neutralizing epitope to obtain information for vaccine design. We found that mAb IGH526 bound to a discontinuous epitope, but with a major component corresponding to E1 residues 314–324. The crystal structure of IGH526 Fab with this E1 glycopeptide at 1.75Å resolution revealed that the antibody binds to one face of an α-helical peptide. Single mutations on the helix substantially lowered IGH526 binding but did not affect neutralization, indicating either that multiple mutations are required or that additional regions are recognized by the antibody in the context of the membrane-associated envelope oligomer. Molecular dynamics simulations indicate that the free peptide is flexible in solution, suggesting that it requires stabilization for use as a candidate vaccine immunogen.
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    Categories: Journal Articles
  • C-terminal threonine reduces Aβ43 amyloidogenicity compared with Aβ42
    [Jul 2015]

    Publication date: Available online 26 June 2015
    Source:Journal of Molecular Biology

    Author(s): Saketh Chemuru, Ravindra Kodali, Ronald Wetzel

    Aβ43, a product of the proteolysis of the amyloid precursor protein APP, is related to Aβ42 by an additional Thr residue at the C-terminus. Aβ43 is typically generated at low levels compared with the predominant Aβ42 and Aβ40 forms, but it has been suggested that this longer peptide might have an impact on Aβ aggregation and Alzheimer’s disease that is out of proportion to its brain content. Here we report that Aβ42 and Aβ43 both spontaneously aggregate into mature amyloid fibrils via sequential appearance of the same series of oligomeric and protofibrillar intermediates, the earliest of which appears to lack β-structure. In spite of the additional β-branched amino acid at the C-terminus, Aβ43 fibrils have fewer strong backbone H-bonds than Aβ42 fibrils, some of which are lost at the C-terminus. In contrast to previous reports, we found that Aβ43 spontaneously aggregates more slowly than Aβ42. In addition, Aβ43 fibrils are very inefficient at seeding Aβ42 amyloid formation, even though Aβ42 fibrils efficiently seed amyloid formation by Aβ43 monomers. Finally, mixtures of Aβ42 and Aβ43 aggregate more slowly than Aβ42 alone. Both in this Aβ42/Aβ43 co-aggregation reaction and in cross-seeding by Aβ42 fibrils, the structure of the Aβ43 in the product fibrils is influenced by the presence of Aβ42. The results provide new details of amyloid structure and assembly pathways, an example of structural plasticity in prion-like replication, and data showing that low levels of Aβ43 in the brain are unlikely to favorably impact the aggregation of Aβ42.





    Categories: Journal Articles
  • Motility, Chemotaxis and Aerotaxis Contribute to Competitiveness during Bacterial Pellicle Biofilm Development
    [Jul 2015]

    Publication date: Available online 26 June 2015
    Source:Journal of Molecular Biology

    Author(s): Theresa Hölscher, Benjamin Bartels, Yu-Cheng Lin, Ramses Gallegos-Monterrosa, Alexa Price-Whelan, Roberto Kolter, Lars E.P. Dietrich, Ákos T. Kovács

    Biofilm formation is a complex process involving various signaling pathways and changes in gene expression. Many of the sensory mechanisms and regulatory cascades involved have been defined for biofilms formed by diverse organisms attached to solid surfaces. By comparison, our knowledge on the basic mechanisms underlying the formation of biofilms at air–liquid interfaces, that is, pellicles, is much less complete. In particular, the roles of flagella have been studied in multiple solid-surface biofilm models but remain largely undefined for pellicles. In this work, we characterize the contributions of flagellum-based motility, chemotaxis and oxygen sensing to pellicle formation in the Gram-positive Bacillus subtilis. We confirm that flagellum-based motility is involved in, but is not absolutely essential for, B. subtilis pellicle formation. Further, we show that flagellum-based motility, chemotaxis and oxygen sensing are important for successful competition during B. subtilis pellicle formation. We report that flagellum-based motility similarly contributes to pellicle formation and fitness in competition assays in the Gram-negative Pseudomonas aeruginosa. Time-lapse imaging of static liquid cultures demonstrates that, in both B. subtilis and P. aeruginosa, a turbulent flow forms in the tube and a zone of clearing appears below the air–liquid interface just before the formation of the pellicle but only in strains that have flagella.
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    Categories: Journal Articles
  • Signaling Control of Differentiation of Embryonic Stem Cells toward Mesendoderm
    [Jul 2015]

    Publication date: Available online 25 June 2015
    Source:Journal of Molecular Biology

    Author(s): Lu Wang, Ye-Guang Chen

    Mesendoderm (ME) refers to the primitive streak in mammalian embryos, which has the ability to further differentiate into mesoderm and endoderm. A better understanding on the regulatory networks of ME differentiation of embryonic stem (ES) cells would provide important insights on early embryo patterning and a possible guidance for ES applications in regenerative medicine. Studies on developmental biology and embryology have offered a great deal of knowledge about key signaling pathways involved in primitive streak formation. Recently, various chemically defined recipes have been formulated to induce differentiation of ES cells toward ME in vitro, which greatly facilitate the elucidation of the regulatory mechanisms of different signals involved in ME specification. Among the extrinsic signals, transforming growth factor-β/Activin signaling and Wnt signaling have been shown to be the most critical ones. On another side, intrinsic epigenetic regulation has been indicated to be important in ME determination. In this review, we summarize the current understanding on the extrinsic and intrinsic regulations of ES cells-to-ME differentiation and the crosstalk among them, aiming to get a general overview on ME specification and primitive streak formation.
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    Categories: Journal Articles
  • ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3′-Azido cDNAs
    [Jul 2015]

    Publication date: Available online 24 June 2015
    Source:Journal of Molecular Biology

    Author(s): Andrew Routh, Steven R. Head, Phillip Ordoukhanian, John E. Johnson

    We present a simple method called “ClickSeq” for NGS (next-generation sequencing) library synthesis that uses click chemistry rather than enzymatic reactions for the ligation of Illumina sequencing adaptors. In ClickSeq, randomly primed reverse transcription reactions are supplemented with azido-2′,3′-dideoxynucleotides that randomly terminate DNA synthesis and release 3′-azido-blocked cDNA fragments in a process akin to dideoxy-Sanger sequencing. Purified fragments are “click ligated” via copper-catalyzed alkyne-azide cycloaddition to DNA oligos modified with a 5′-alkyne group. This generates ssDNA molecules containing an unnatural triazole-linked DNA backbone that is sufficiently biocompatible for PCR amplification to generate a cDNA library for RNAseq. Here, we analyze viral RNAs and mRNA to demonstrate that ClickSeq produces unbiased NGS libraries with low error rates comparable to standard methods. Importantly, ClickSeq is robust against common artifacts of NGS such as chimera formation and artifactual recombination with fewer than 3 aberrant events detected per million reads.
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    Categories: Journal Articles