Publications by authors named "James A J Fitzpatrick"

51 Publications

Liver specific deletion of mouse Tm6sf2 promotes steatosis, fibrosis and hepatocellular cancer.

Hepatology 2021 Feb 27. Epub 2021 Feb 27.

Department of Medicine, Washington University School of Medicine, St. Louis, MO, 63110, United States.

Background And Aims: Human TM6SF2 variant rs58542926 is associated with nonalcoholic fatty liver disease (NAFLD) and hepatocellular cancer (HCC). However, conflicting reports in germline Tm6sf2 knockout mice suggest no change or decreased VLDL secretion and either unchanged or increased hepatic steatosis, with no increased fibrosis. We generated liver specific Tm6Sf2 knockout mice (Tm6 LKO) to study VLDL secretion and the impact on development and progression of NAFLD.

Approach And Results: Two independent lines of Tm6 LKO mice exhibited spontaneous hepatic steatosis. Targeted lipidomic analyses showed increased triglyceride (TG) species whose distribution and abundance phenocopied findings in mice with liver specific deletion of microsomal triglyceride transfer protein. VLDL TG secretion was reduced, with small, underlipidated particles and unchanged or increased APOB. Liver-specific adeno-associated viral (AAV8-TBG) rescue using either wild type (WT) or mutant E167K-Tm6 reduced hepatic steatosis and improved VLDL secretion. Tm6 LKO mice fed a high milk-fat diet for 3 weeks exhibited increased steatosis and fibrosis and those phenotypes were further exacerbated when mice were fed fibrogenic, high fat/fructose diets for 20 weeks. In two models of HCC, either neonatal mice injected with streptozotocin (NASH/STAM) and high fat fed or with diethylnitrosamine (DEN) injection plus fibrogenic diet feeding, Tm6 LKO mice exhibited increased steatosis, greater tumor burden and increased tumor area versus Tm6 flox controls. Additionally, DEN-injected and fibrogenic diet fed Tm6 LKO mice administered WT Tm6 or E167K-mutant Tm6 AAV8 revealed significant tumor attenuation, with tumor burden inversely correlated with Tm6 protein levels.

Conclusions: Liver-specific Tm6sf2 deletion impairs VLDL secretion, promoting hepatic steatosis, fibrosis and accelerated development of HCC, which was mitigated with AAV8- mediated rescue.
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http://dx.doi.org/10.1002/hep.31771DOI Listing
February 2021

Cryo-EM structure of a proton-activated chloride channel TMEM206.

Sci Adv 2021 Feb 24;7(9). Epub 2021 Feb 24.

Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, MO 63110, USA.

TMEM206 has been recently identified as an evolutionarily conserved chloride channel that underlies ubiquitously expressed, proton-activated, outwardly rectifying anion currents. Here, we report the cryo-electron microscopy structure of pufferfish TMEM206, which forms a trimeric channel, with each subunit comprising two transmembrane segments and a large extracellular domain. An ample vestibule in the extracellular region is accessible laterally from the three side portals. The central pore contains multiple constrictions. A conserved lysine residue near the cytoplasmic end of the inner helix forms the presumed chloride ion selectivity filter. Unprecedentedly, the core structure and assembly closely resemble those of the epithelial sodium channel/degenerin family of sodium channels that are unrelated in amino acid sequence and conduct cations instead of anions. Together with electrophysiology, this work provides insights into ion conduction and gating for a new class of chloride channels that is architecturally distinct from previously characterized chloride channel families.
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http://dx.doi.org/10.1126/sciadv.abe5983DOI Listing
February 2021

Size-Dependent Effective Diffusivity in Healthy Human and Porcine Joint Synovium.

Ann Biomed Eng 2021 Jan 25. Epub 2021 Jan 25.

Department of Biomedical Engineering, Washington University in St. Louis, 190 Whitaker Hall, 1 Brookings Drive, St. Louis, MO, 63130, USA.

Intra-articular drug delivery can be effective in targeting a diseased joint but is hampered by rapid clearance times from the diarthrodial joint. The synovium is a multi-layered tissue that surrounds the diarthrodial joint and governs molecular transport into and out of the joint. No models of drug clearance through synovium exist to quantify diffusivity across solutes, tissue type and disease pathology. We previously have developed a finite element model of synovium as a porous, permeable, fluid-filled tissue and used an inverse method to determine urea's effective diffusivity (D) in de-vitalized synovium explants. Here we apply this method to determine D from unsteady diffusive transport of model solutes and confirm the role of molecular weight in solute transport. As molecular weight increased, D decreased in both human and porcine tissues, with similar behavior across the two species. Unsteady transport was well-described by a single exponential transient decay in concentration, yielding solute half-lives (t) that compared favorably with the D determined from the finite element model fit. Determined values for D parallel prior observations of size-dependent in vivo drug clearance and provide an intrinsic parameter with greater ability to resolve size-dependence in vitro. Thus, this work forms the basis for understanding the influence of size on drug transport in synovium and can guide future studies to elucidate the role of charge and tissue pathology on the transport of therapeutics in healthy and pathological human synovium.
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http://dx.doi.org/10.1007/s10439-020-02717-4DOI Listing
January 2021

Pancreatic β-Cell-Specific Deletion of VPS41 Causes Diabetes Due to Defects in Insulin Secretion.

Diabetes 2021 Feb 9;70(2):436-448. Epub 2020 Nov 9.

Department of Biological Sciences, University of Denver, Denver, CO

Insulin secretory granules (SGs) mediate the regulated secretion of insulin, which is essential for glucose homeostasis. The basic machinery responsible for this regulated exocytosis consists of specific proteins present both at the plasma membrane and on insulin SGs. The protein composition of insulin SGs thus dictates their release properties, yet the mechanisms controlling insulin SG formation, which determine this molecular composition, remain poorly understood. VPS41, a component of the endolysosomal tethering homotypic fusion and vacuole protein sorting (HOPS) complex, was recently identified as a cytosolic factor involved in the formation of neuroendocrine and neuronal granules. We now find that VPS41 is required for insulin SG biogenesis and regulated insulin secretion. Loss of VPS41 in pancreatic β-cells leads to a reduction in insulin SG number, changes in their transmembrane protein composition, and defects in granule-regulated exocytosis. Exploring a human point mutation, identified in patients with neurological but no endocrine defects, we show that the effect on SG formation is independent of HOPS complex formation. Finally, we report that mice with a deletion of VPS41 specifically in β-cells develop diabetes due to severe depletion of insulin SG content and a defect in insulin secretion. In sum, our data demonstrate that VPS41 contributes to glucose homeostasis and metabolism.
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http://dx.doi.org/10.2337/db20-0454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881869PMC
February 2021

Assaying three-dimensional cellular architecture using X-ray tomographic and correlated imaging approaches.

J Biol Chem 2020 11 16;295(46):15782-15793. Epub 2020 Sep 16.

Washington University Center for Cellular Imaging, Washington University School of Medicine, Saint Louis, Missouri, USA

Much of our understanding of the spatial organization of and interactions between cellular organelles and macromolecular complexes has been the result of imaging studies utilizing either light- or electron-based microscopic analyses. These classical approaches, while insightful, are nonetheless limited either by restrictions in resolution or by the sheer complexity of generating multidimensional data. Recent advances in the use and application of X-rays to acquire micro- and nanotomographic data sets offer an alternative methodology to visualize cellular architecture at the nanoscale. These new approaches allow for the subcellular analyses of unstained vitrified cells and three-dimensional localization of specific protein targets and have served as an essential tool in bridging light and electron correlative microscopy experiments. Here, we review the theory, instrumentation details, acquisition principles, and applications of both soft X-ray tomography and X-ray microscopy and how the use of these techniques offers a succinct means of analyzing three-dimensional cellular architecture. We discuss some of the recent work that has taken advantage of these approaches and detail how they have become integral in correlative microscopy workflows.
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http://dx.doi.org/10.1074/jbc.REV120.009633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667966PMC
November 2020

Structural mechanism for gating of a eukaryotic mechanosensitive channel of small conductance.

Nat Commun 2020 07 23;11(1):3690. Epub 2020 Jul 23.

Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, MO, 63110, USA.

Mechanosensitive ion channels transduce physical force into electrochemical signaling that underlies an array of fundamental physiological processes, including hearing, touch, proprioception, osmoregulation, and morphogenesis. The mechanosensitive channels of small conductance (MscS) constitute a remarkably diverse superfamily of channels critical for management of osmotic pressure. Here, we present cryo-electron microscopy structures of a MscS homolog from Arabidopsis thaliana, MSL1, presumably in both the closed and open states. The heptameric MSL1 channel contains an unusual bowl-shaped transmembrane region, which is reminiscent of the evolutionarily and architecturally unrelated mechanosensitive Piezo channels. Upon channel opening, the curved transmembrane domain of MSL1 flattens and expands. Our structures, in combination with functional analyses, delineate a structural mechanism by which mechanosensitive channels open under increased membrane tension. Further, the shared structural feature between unrelated channels suggests the possibility of a unified mechanical gating mechanism stemming from membrane deformation induced by a non-planar transmembrane domain.
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http://dx.doi.org/10.1038/s41467-020-17538-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378837PMC
July 2020

Gating of human TRPV3 in a lipid bilayer.

Nat Struct Mol Biol 2020 07 22;27(7):635-644. Epub 2020 Jun 22.

Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, MO, USA.

The transient receptor potential cation channel subfamily V member 3 (TRPV3) channel plays a critical role in skin physiology, and mutations in TRPV3 result in the development of a congenital skin disorder, Olmsted syndrome. Here we describe multiple cryo-electron microscopy structures of human TRPV3 reconstituted into lipid nanodiscs, representing distinct functional states during the gating cycle. The ligand-free, closed conformation reveals well-ordered lipids interacting with the channel and two physical constrictions along the ion-conduction pore involving both the extracellular selectivity filter and intracellular helix bundle crossing. Both the selectivity filter and bundle crossing expand upon activation, accompanied by substantial structural rearrangements at the cytoplasmic intersubunit interface. Transition to the inactivated state involves a secondary structure change of the pore-lining helix, which contains a π-helical segment in the closed and open conformations, but becomes entirely α-helical upon inactivation. Together with electrophysiological characterization, structures of TRPV3 in a lipid membrane environment provide unique insights into channel activation and inactivation mechanisms.
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http://dx.doi.org/10.1038/s41594-020-0428-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354234PMC
July 2020

Cryo-EM structures of the ATP release channel pannexin 1.

Nat Struct Mol Biol 2020 04 30;27(4):373-381. Epub 2020 Mar 30.

Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, USA.

The plasma membrane adenosine triphosphate (ATP) release channel pannexin 1 (PANX1) has been implicated in many physiological and pathophysiological processes associated with purinergic signaling, including cancer progression, apoptotic cell clearance, inflammation, blood pressure regulation, oocyte development, epilepsy and neuropathic pain. Here we present near-atomic-resolution structures of human and frog PANX1 determined by cryo-electron microscopy that revealed a heptameric channel architecture. Compatible with ATP permeation, the transmembrane pore and cytoplasmic vestibule were exceptionally wide. An extracellular tryptophan ring located at the outer pore created a constriction site, potentially functioning as a molecular sieve that restricts the size of permeable substrates. The amino and carboxyl termini, not resolved in the density map, appeared to be structurally dynamic and might contribute to narrowing of the pore during channel gating. In combination with functional characterization, this work elucidates the previously unknown architecture of pannexin channels and establishes a foundation for understanding their unique channel properties.
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http://dx.doi.org/10.1038/s41594-020-0401-0DOI Listing
April 2020

Cryo-EM structure of a neuronal functional amyloid implicated in memory persistence in .

Science 2020 03;367(6483):1230-1234

Stowers Institute for Medical Research, Kansas City, MO 64110, USA.

How long-lived memories withstand molecular turnover is a fundamental question. Aggregates of a prion-like RNA-binding protein, cytoplasmic polyadenylation element-binding (CPEB) protein, is a putative substrate of long-lasting memories. We isolated aggregated CPEB, Orb2, from adult heads and determined its activity and atomic structure, at 2.6-angstrom resolution, using cryo-electron microscopy. Orb2 formed ~75-nanometer-long threefold-symmetric amyloid filaments. Filament formation transformed Orb2 from a translation repressor to an activator and "seed" for further translationally active aggregation. The 31-amino acid protofilament core adopted a cross-β unit with a single hydrophilic hairpin stabilized through interdigitated glutamine packing. Unlike the hydrophobic core of pathogenic amyloids, the hydrophilic core of Orb2 filaments suggests how some neuronal amyloids could be a stable yet regulatable substrate of memory.
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http://dx.doi.org/10.1126/science.aba3526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7182444PMC
March 2020

Staphylococcus aureus Infects Osteoclasts and Replicates Intracellularly.

mBio 2019 10 15;10(5). Epub 2019 Oct 15.

Division of Bone & Mineral Diseases, Musculoskeletal Research Center, Washington University School of Medicine, Saint Louis, Missouri, USA

Osteomyelitis (OM), or inflammation of bone tissue, occurs most frequently as a result of bacterial infection and severely perturbs bone structure. OM is predominantly caused by , and even with proper treatment, OM has a high rate of recurrence and chronicity. While has been shown to infect osteoblasts, it remains unclear whether osteoclasts (OCs) are also a target of intracellular infection. Here, we demonstrate the ability of to intracellularly infect and divide within OCs. OCs were differentiated from bone marrow macrophages (BMMs) by exposure to receptor activator of nuclear factor kappa-B ligand (RANKL). By utilizing an intracellular survival assay and flow cytometry, we found that at 18 h postinfection the intracellular burden of increased dramatically in cells with at least 2 days of RANKL exposure, while the bacterial burden decreased in BMMs. To further explore the signals downstream of RANKL, we manipulated factors controlling OC differentiation, NFATc1 and alternative NF-κB, and found that intracellular bacterial growth correlates with NFATc1 levels in RANKL-treated cells. Confocal and time-lapse microscopy in mature OCs showed a range of intracellular infection that correlated inversely with -phagolysosome colocalization. The propensity of OCs to become infected, paired with their diminished bactericidal capacity compared to BMMs, could promote OM progression by allowing to evade initial immune regulation and proliferate at the periphery of lesions where OCs are most abundant. The inflammation of bone tissue is called osteomyelitis, and most cases are caused by an infection with the bacterium To date, the bone-building cells, osteoblasts, have been implicated in the progression of these infections, but not much is known about how the bone-resorbing cells, osteoclasts, participate. In this study, we show that can infect osteoclasts and proliferate inside these cells, whereas bone-residing macrophages, immune cells related to osteoclasts, destroy the bacteria. These findings elucidate a unique role for osteoclasts to harbor bacteria during infection, providing a possible mechanism by which bacteria could evade destruction by the immune system.
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http://dx.doi.org/10.1128/mBio.02447-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794488PMC
October 2019

Combined Experimental Approach and Finite Element Modeling of Small Molecule Transport Through Joint Synovium to Measure Effective Diffusivity.

J Biomech Eng 2019 Sep 1. Epub 2019 Sep 1.

Department of Biomedical Engineering, Washington University in St. Louis, Whitaker Hall, 1 Brookings Dr., St. Louis, MO, 63130.

Trans-synovial solute transport plays a critical role in the clearance of intra-articularly delivered drugs. In this study, we present a computational finite element model of solute transport through the synovium validated by experiments on synovial explants. Unsteady diffusion of urea, a small uncharged molecule, was measured through devitalized porcine and human synovium using custom-built diffusion chambers. A multiphasic computational model was constructed and optimized with the experimental data to extract effective diffusivity for urea within the synovium. A monotonic decrease in urea concentration was observed in the donor bath over time, with an effective diffusivity found to be an order of magnitude lower in synovium versus that measured in free solution. Parametric studies incorporating an intimal cell layer with varying thickness and varying effective diffusivities were performed, revealing a dependence of drug clearance kinetics on both parameters. The findings of this study indicate that the synovial matrix impedes urea solute transport out of the joint with little retention of the solute in the matrix.
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http://dx.doi.org/10.1115/1.4044892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104772PMC
September 2019

Tmem178 negatively regulates store-operated calcium entry in myeloid cells via association with STIM1.

J Autoimmun 2019 07 22;101:94-108. Epub 2019 Apr 22.

Department of Orthopaedics, Washington University School of Medicine, St. Louis, MO, 63110, USA; Shriners Hospitals for Children, St. Louis MO, USA. Electronic address:

Store-operated calcium entry (SOCE) modulates cytosolic calcium in multiple cells. Endoplasmic reticulum (ER)-localized STIM1 and plasma membrane (PM)-localized ORAI1 are two main components of SOCE. STIM1:ORAI1 association requires STIM1 oligomerization, its re-distribution to ER-PM junctions, and puncta formation. However, little is known about the negative regulation of these steps to prevent calcium overload. Here, we identified Tmem178 as a negative modulator of STIM1 puncta formation in myeloid cells. Using site-directed mutagenesis, co-immunoprecipitation assays and FRET imaging, we determined that Tmem178:STIM1 association occurs via their transmembrane motifs. Mutants that increase Tmem178:STIM1 association reduce STIM1 puncta formation, SOCE activation, impair inflammatory cytokine production in macrophages and osteoclastogenesis. Mutants that reduce Tmem178:STIM1 association reverse these effects. Furthermore, exposure to plasma from arthritic patients decreases Tmem178 expression, enhances SOCE activation and cytoplasmic calcium. In conclusion, Tmem178 modulates the rate-limiting step of STIM1 puncta formation and therefore controls SOCE in inflammatory conditions.
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http://dx.doi.org/10.1016/j.jaut.2019.04.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7102427PMC
July 2019

Vesicular Glutamatergic Transmission in Noise-Induced Loss and Repair of Cochlear Ribbon Synapses.

J Neurosci 2019 06 29;39(23):4434-4447. Epub 2019 Mar 29.

Department of Otolaryngology, Washington University School of Medicine,

Noise-induced excitotoxicity is thought to depend on glutamate. However, the excitotoxic mechanisms are unknown, and the necessity of glutamate for synapse loss or regeneration is unclear. Despite absence of glutamatergic transmission from cochlear inner hair cells in mice lacking the vesicular glutamate transporter-3 ( ), at 9-11 weeks, approximately half the number of synapses found in were maintained as postsynaptic AMPA receptors juxtaposed with presynaptic ribbons and voltage-gated calcium channels (Ca1.3). Synapses were larger in than In and mice, 8-16 kHz octave-band noise exposure at 100 dB sound pressure level caused a threshold shift (∼40 dB) and a loss of synapses (>50%) at 24 h after exposure. Hearing threshold and synapse number partially recovered by 2 weeks after exposure as ribbons became larger, whereas recovery was significantly better in Noise exposure at 94 dB sound pressure level caused auditory threshold shifts that fully recovered in 2 weeks, whereas suprathreshold hearing recovered faster in than These results, from mice of both sexes, suggest that spontaneous repair of synapses after noise depends on the level of Vglut3 protein or the level of glutamate release during the recovery period. Noise-induced loss of presynaptic ribbons or postsynaptic AMPA receptors was not observed in , demonstrating its dependence on vesicular glutamate release. In and , noise exposure caused unpairing of presynaptic ribbons and presynaptic Ca1.3, but not in where Ca1.3 remained clustered with ribbons at presynaptic active zones. These results suggest that, without glutamate release, noise-induced presynaptic Ca influx was insufficient to disassemble the active zone. However, synapse volume increased by 2 weeks after exposure in , suggesting glutamate-independent mechanisms. Hearing depends on glutamatergic transmission mediated by Vglut3, but the role of glutamate in synapse loss and repair is unclear. Here, using mice of both sexes, we show that one copy of the gene is sufficient for noise-induced threshold shift and loss of ribbon synapses, but both copies are required for normal recovery of hearing function and ribbon synapse number. Impairment of the recovery process in mice having only one functional copy suggests that glutamate release may promote synapse regeneration. At least one copy of the gene is necessary for noise-induced synapse loss. Although the excitotoxic mechanism remains unknown, these findings are consistent with the presumption that glutamate is the key mediator of noise-induced synaptopathy.
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http://dx.doi.org/10.1523/JNEUROSCI.2228-18.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6554621PMC
June 2019

Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes.

PLoS One 2018 21;13(12):e0209201. Epub 2018 Dec 21.

Department of Pediatrics, Divisions of Critical Care Medicine, Washington University in Saint Louis, School of Medicine, Saint Louis, MO, United States of America.

Intact red blood cells (RBCs) are required for phenotypic analyses. In order to allow separation (time and location) between subject encounter and sample analysis, we developed a research-specific RBC cryopreservation protocol and assessed its impact on data fidelity for key biochemical and physiological assays. RBCs drawn from healthy volunteers were aliquotted for immediate analysis or following glycerol-based cryopreservation, thawing, and deglycerolization. RBC phenotype was assessed by (1) scanning electron microscopy (SEM) imaging and standard morphometric RBC indices, (2) osmotic fragility, (3) deformability, (4) endothelial adhesion, (5) oxygen (O2) affinity, (6) ability to regulate hypoxic vasodilation, (7) nitric oxide (NO) content, (8) metabolomic phenotyping (at steady state, tracing with [1,2,3-13C3]glucose ± oxidative challenge with superoxide thermal source; SOTS-1), as well as in vivo quantification (following human to mouse RBC xenotransfusion) of (9) blood oxygenation content mapping and flow dynamics (velocity and adhesion). Our revised glycerolization protocol (40% v/v final) resulted in >98.5% RBC recovery following freezing (-80°C) and thawing (37°C), with no difference compared to the standard reported method (40% w/v final). Full deglycerolization (>99.9% glycerol removal) of 40% v/v final samples resulted in total cumulative lysis of ~8%, compared to ~12-15% with the standard method. The post cryopreservation/deglycerolization RBC phenotype was indistinguishable from that for fresh RBCs with regard to physical RBC parameters (morphology, volume, and density), osmotic fragility, deformability, endothelial adhesivity, O2 affinity, vasoregulation, metabolomics, and flow dynamics. These results indicate that RBC cryopreservation/deglycerolization in 40% v/v glycerol final does not significantly impact RBC phenotype (compared to fresh cells).
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0209201PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303082PMC
May 2019

Compound C inhibits nonsense-mediated RNA decay independently of AMPK.

PLoS One 2018 5;13(10):e0204978. Epub 2018 Oct 5.

Department of Cell Biology & Physiology, Washington University School of Medicine, St. Louis, Missouri, United States of America.

The nonsense mediated RNA decay (NMD) pathway safeguards the integrity of the transcriptome by targeting mRNAs with premature translation termination codons (PTCs) for degradation. It also regulates gene expression by degrading a large number of non-mutant RNAs (including mRNAs and noncoding RNAs) that bear NMD-inducing features. Consequently, NMD has been shown to influence development, cellular response to stress, and clinical outcome of many genetic diseases. Small molecules that can modulate NMD activity provide critical tools for understanding the mechanism and physiological functions of NMD, and they also offer potential means for treating certain genetic diseases and cancer. Therefore, there is an intense interest in identifying small-molecule NMD inhibitors or enhancers. It was previously reported that both inhibition of NMD and treatment with the AMPK-selective inhibitor Compound C (CC) induce autophagy in human cells, raising the possibility that CC may be capable of inhibiting NMD. Here we show that CC indeed has a NMD-inhibitory activity. Inhibition of NMD by CC is, however, independent of AMPK activity. As a competitive ATP analog, CC does not affect the kinase activity of SMG1, an essential NMD factor and the only known kinase in the NMD pathway. However, CC treatment down-regulates the protein levels of several NMD factors. The induction of autophagy by CC treatment is independent of ATF4, a NMD target that has been shown to promote autophagy in response to NMD inhibition. Our results reveal a new activity of CC as a NMD inhibitor, which has implications for its use in basic research and drug development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0204978PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173407PMC
March 2019

The Zinc Finger Transcription Factor PLAGL2 Enhances Stem Cell Fate and Activates Expression of ASCL2 in Intestinal Epithelial Cells.

Stem Cell Reports 2018 08 12;11(2):410-424. Epub 2018 Jul 12.

Department of Medicine, Division of Gastroenterology, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8124, CSRB NT 923, Saint Louis, MO 63110, USA. Electronic address:

Intestinal epithelial stem cell (IESC) fate is promoted by two major transcriptional regulators, the TCF4/β-catenin complex and ASCL2, which drive expression of IESC-specific factors, including Lgr5, Ephb2, and Rnf43. Canonical Wnt signaling via TCF4/β-catenin directly transactivates Ascl2, which in turn auto-regulates its own expression. Conversely, Let-7 microRNAs antagonize the IESC lineage by repressing specific mRNA targets. Here, we identify the zinc finger transcription factor PLAGL2 as a Let-7 target that regulates IESC fate. PLAGL2 drives an IESC expression signature, activates Wnt gene expression, and enhances a TCF/LEF reporter in intestinal organoids. In parallel, via cell-autonomous mechanisms, PLAGL2 is required for lineage clonal expansion and directly enhances expression of ASCL2. PLAGL2 also supports enteroid growth and survival in the context of Wnt ligand depletion. PLAGL2 expression is strongly associated with an IESC signature in colorectal cancer and may be responsible for contributing to the aberrant activation of an immature phenotype.
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http://dx.doi.org/10.1016/j.stemcr.2018.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6092695PMC
August 2018

Modern Laser Scanning Confocal Microscopy.

Curr Protoc Cytom 2018 07 20;85(1):e39. Epub 2018 Jun 20.

Center for Cellular Imaging, Washington University in St. Louis, St. Louis, Missouri.

Since its commercialization in the late 1980's, confocal laser scanning microscopy (CLSM) has since become one of the most prevalent fluorescence microscopy techniques for three-dimensional structural studies of biological cells and tissues. The flexibility of the approach has enabled its application in a diverse array of studies, from the fast imaging of dynamic processes in living cells, to meticulous morphological analyses of tissues, and co-localization of protein expression patterns. In this chapter, we introduce the principles of confocal microscopy and discuss how the approach has become a mainstay in the biological sciences. We describe the components of a CLSM system and assess how modern implementations of the approach have further expanded the use of the technique. Finally, we briefly outline some practical considerations to take into account when acquiring data using a CLSM system. © 2018 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpcy.39DOI Listing
July 2018

Characterization of the bone marrow adipocyte niche with three-dimensional electron microscopy.

Bone 2019 01 31;118:89-98. Epub 2018 Jan 31.

Division of Bone and Mineral Diseases, Department of Medicine, Washington University, St. Louis, MO, USA; Department of Cell Biology & Physiology, Washington University, St. Louis, MO, USA. Electronic address:

Unlike white and brown adipose tissues, the bone marrow adipocyte (BMA) exists in a microenvironment containing unique populations of hematopoietic and skeletal cells. To study this microenvironment at the sub-cellular level, we performed a three-dimensional analysis of the ultrastructure of the BMA niche with focused ion beam scanning electron microscopy (FIB-SEM). This revealed that BMAs display hallmarks of metabolically active cells including polarized lipid deposits, a dense mitochondrial network, and areas of endoplasmic reticulum. The distinct orientations of the triacylglycerol droplets suggest that fatty acids are taken up and/or released in three key areas - at the endothelial interface, into the hematopoietic milieu, and at the bone surface. Near the sinusoidal vasculature, endothelial cells send finger-like projections into the surface of the BMA which terminate near regions of lipid within the BMA cytoplasm. In some regions, perivascular cells encase the BMA with their flattened cellular projections, limiting contacts with other cells in the niche. In the hematopoietic milieu, BMAT adipocytes of the proximal tibia interact extensively with maturing cells of the myeloid/granulocyte lineage. Associations with erythroblast islands are also prominent. At the bone surface, the BMA extends organelle and lipid-rich cytoplasmic regions toward areas of active osteoblasts. This suggests that the BMA may serve to partition nutrient utilization between diverse cellular compartments, serving as an energy-rich hub of the stromal-reticular network. Lastly, though immuno-EM, we've identified a subset of bone marrow adipocytes that are innervated by the sympathetic nervous system, providing an additional mechanism for regulation of the BMA. In summary, this work reveals that the bone marrow adipocyte is a dynamic cell with substantial capacity for interactions with the diverse components of its surrounding microenvironment. These local interactions likely contribute to its unique regulation relative to peripheral adipose tissues.
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http://dx.doi.org/10.1016/j.bone.2018.01.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063802PMC
January 2019

Reciprocal Spatiotemporally Controlled Apoptosis Regulates Wolffian Duct Cloaca Fusion.

J Am Soc Nephrol 2018 03 11;29(3):775-783. Epub 2018 Jan 11.

Division of Nephrology, Department of Internal Medicine,

The epithelial Wolffian duct (WD) inserts into the cloaca (primitive bladder) before metanephric kidney development, thereby establishing the initial plumbing for eventual joining of the ureters and bladder. Defects in this process cause common anomalies in the spectrum of congenital anomalies of the kidney and urinary tract (CAKUT). However, developmental, cellular, and molecular mechanisms of WD-cloaca fusion are poorly understood. Through systematic analysis of early WD tip development in mice, we discovered that a novel process of spatiotemporally regulated apoptosis in WD and cloaca was necessary for WD-cloaca fusion. Aberrant RET tyrosine kinase signaling through tyrosine (Y) 1062, to which PI3K- or ERK-activating proteins dock, or Y1015, to which PLC docks, has been shown to cause CAKUT-like defects. Cloacal apoptosis did not occur in RetY1062F mutants, in which WDs did not reach the cloaca, or in RetY1015F mutants, in which WD tips reached the cloaca but did not fuse. Moreover, inhibition of ERK or apoptosis prevented WD-cloaca fusion in cultures, and WD-specific genetic deletion of YAP attenuated cloacal apoptosis and WD-cloacal fusion Thus, cloacal apoptosis requires direct contact and signals from the WD tip and is necessary for WD-cloacal fusion. These findings may explain the mechanisms of many CAKUT.
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http://dx.doi.org/10.1681/ASN.2017040380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827592PMC
March 2018

Nonlinear optical imaging of extracellular matrix proteins.

Methods Cell Biol 2018 28;143:57-78. Epub 2017 Oct 28.

Washington University Center for Cellular Imaging, Washington University School of Medicine, St. Louis, MO, United States. Electronic address:

Over the last 2 decades, nonlinear imaging methods such as multiharmonic imaging microscopy (MHIM) have become powerful approaches for the label-free visualization of biological structures. Multiharmonic signals are generated when an intense electromagnetic field propagates through a sample that either has a specific molecular orientation or exhibits certain physical properties. It can provide complementary morphological information when integrated with other nonlinear optical imaging techniques such as two-photon excitation (TPE). Here, we present the necessary methodology to implement an integrated approach for multiharmonic and TPE imaging of the mouse aorta using a commercial two-photon microscope. This approach illustrates how to differentiate the microstructure of the mouse aorta that are due to collagen fibrils and elastic laminae under 820 and 1230nm excitation. Our method also demonstrates how to perform multiharmonic generation by reflectance of the forwardly propagating emission signal. The ability to visualize biological samples without additional genetically targeted or chemical stains makes MHIM well suited for studying the morphology of the mouse aorta and has the potential to be applied to other collagen and elastin-rich tissues.
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http://dx.doi.org/10.1016/bs.mcb.2017.08.004DOI Listing
November 2018

HID-1 controls formation of large dense core vesicles by influencing cargo sorting and -Golgi network acidification.

Mol Biol Cell 2017 Dec 26;28(26):3870-3880. Epub 2017 Oct 26.

Department of Biological Sciences, University of Denver, Denver, CO 80210

Large dense core vesicles (LDCVs) mediate the regulated release of neuropeptides and peptide hormones. They form at the -Golgi network (TGN), where their soluble content aggregates to form a dense core, but the mechanisms controlling biogenesis are still not completely understood. Recent studies have implicated the peripheral membrane protein HID-1 in neuropeptide sorting and insulin secretion. Using CRISPR/Cas9, we generated HID-1 KO rat neuroendocrine cells, and we show that the absence of HID-1 results in specific defects in peptide hormone and monoamine storage and regulated secretion. Loss of HID-1 causes a reduction in the number of LDCVs and affects their morphology and biochemical properties, due to impaired cargo sorting and dense core formation. HID-1 KO cells also exhibit defects in TGN acidification together with mislocalization of the Golgi-enriched vacuolar H-ATPase subunit isoform a2. We propose that HID-1 influences early steps in LDCV formation by controlling dense core formation at the TGN.
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http://dx.doi.org/10.1091/mbc.E17-08-0491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5739301PMC
December 2017

Catheterization alters bladder ecology to potentiate infection of the urinary tract.

Proc Natl Acad Sci U S A 2017 10 25;114(41):E8721-E8730. Epub 2017 Sep 25.

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110;

Methicillin-resistant (MRSA) is an emerging cause of catheter-associated urinary tract infection (CAUTI), which frequently progresses to more serious invasive infections. We adapted a mouse model of CAUTI to investigate how catheterization increases an individual's susceptibility to MRSA UTI. This analysis revealed that catheterization was required for MRSA to achieve high-level, persistent infection in the bladder. As shown previously, catheter placement induced an inflammatory response resulting in the release of the host protein fibrinogen (Fg), which coated the bladder and implant. Following infection, we showed that MRSA attached to the urothelium and implant in patterns that colocalized with deposited Fg. Furthermore, MRSA exacerbated the host inflammatory response to stimulate the additional release and accumulation of Fg in the urinary tract, which facilitated MRSA colonization. Consistent with this model, analysis of catheters from patients with -positive cultures revealed colocalization of Fg, which was deposited on the catheter, with Clumping Factors A and B (ClfA and ClfB) have been shown to contribute to MRSA-Fg interactions in other models of disease. We found that mutants in had significantly greater Fg-binding defects than mutants in in several in vitro assays. Paradoxically, only the ClfB strain was significantly attenuated in the CAUTI model. Together, these data suggest that catheterization alters the urinary tract environment to promote MRSA CAUTI pathogenesis by inducing the release of Fg, which the pathogen enhances to persist in the urinary tract despite the host's robust immune response.
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http://dx.doi.org/10.1073/pnas.1707572114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5642702PMC
October 2017

Bone-Induced Expression of Integrin β3 Enables Targeted Nanotherapy of Breast Cancer Metastases.

Cancer Res 2017 11 30;77(22):6299-6312. Epub 2017 Aug 30.

Department of Medicine, Division of Molecular Oncology, Washington University School of Medicine, St. Louis, Missouri.

Bone metastases occur in approximately 70% of metastatic breast cancer patients, often leading to skeletal injuries. Current treatments are mainly palliative and underscore the unmet clinical need for improved therapies. In this study, we provide preclinical evidence for an antimetastatic therapy based on targeting integrin β3 (β3), which is selectively induced on breast cancer cells in bone by the local bone microenvironment. In a preclinical model of breast cancer, β3 was strongly expressed on bone metastatic cancer cells, but not primary mammary tumors or visceral metastases. In tumor tissue from breast cancer patients, β3 was significantly elevated on bone metastases relative to primary tumors from the same patient ( = 42). Mechanistic investigations revealed that TGFβ signaling through SMAD2/SMAD3 was necessary for breast cancer induction of β3 within the bone. Using a micelle-based nanoparticle therapy that recognizes integrin αvβ3 (αvβ3-MPs of ∼12.5 nm), we demonstrated specific localization to breast cancer bone metastases in mice. Using this system for targeted delivery of the chemotherapeutic docetaxel, we showed that bone tumor burden could be reduced significantly with less bone destruction and less hepatotoxicity compared with equimolar doses of free docetaxel. Furthermore, mice treated with αvβ3-MP-docetaxel exhibited a significant decrease in bone-residing tumor cell proliferation compared with free docetaxel. Taken together, our results offer preclinical proof of concept for a method to enhance delivery of chemotherapeutics to breast cancer cells within the bone by exploiting their selective expression of integrin αvβ3 at that metastatic site. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-1225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841166PMC
November 2017

A single transcription factor is sufficient to induce and maintain secretory cell architecture.

Genes Dev 2017 01 7;31(2):154-171. Epub 2017 Feb 7.

Division of Gastroenterology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

We hypothesized that basic helix-loop-helix (bHLH) MIST1 (BHLHA15) is a "scaling factor" that universally establishes secretory morphology in cells that perform regulated secretion. Here, we show that targeted deletion of MIST1 caused dismantling of the secretory apparatus of diverse exocrine cells. Parietal cells (PCs), whose function is to pump acid into the stomach, normally lack MIST1 and do not perform regulated secretion. Forced expression of MIST1 in PCs caused them to expand their apical cytoplasm, rearrange mitochondrial/lysosome trafficking, and generate large secretory granules. induced a cohort of genes regulated by MIST1 in multiple organs but did not affect PC function. MIST1 bound CATATG/CAGCTG E boxes in the first intron of genes that regulate autophagosome/lysosomal degradation, mitochondrial trafficking, and amino acid metabolism. Similar alterations in cell architecture and gene expression were also caused by ectopically inducing MIST1 in vivo in hepatocytes. Thus, MIST1 is a scaling factor necessary and sufficient by itself to induce and maintain secretory cell architecture. Our results indicate that, whereas mature cell types in each organ may have unique developmental origins, cells performing similar physiological functions throughout the body share similar transcription factor-mediated architectural "blueprints."
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http://dx.doi.org/10.1101/gad.285684.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322730PMC
January 2017

In vitro myelin formation using embryonic stem cells.

Development 2015 Jun 26;142(12):2213-25. Epub 2015 May 26.

Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA

Myelination in the central nervous system is the process by which oligodendrocytes form myelin sheaths around the axons of neurons. Myelination enables neurons to transmit information more quickly and more efficiently and allows for more complex brain functions; yet, remarkably, the underlying mechanism by which myelination occurs is still not fully understood. A reliable in vitro assay is essential to dissect oligodendrocyte and myelin biology. Hence, we developed a protocol to generate myelinating oligodendrocytes from mouse embryonic stem cells and established a myelin formation assay with embryonic stem cell-derived neurons in microfluidic devices. Myelin formation was quantified using a custom semi-automated method that is suitable for larger scale analysis. Finally, early myelination was followed in real time over several days and the results have led us to propose a new model for myelin formation.
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http://dx.doi.org/10.1242/dev.116517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4483765PMC
June 2015

Neural Mechanisms for Evaluating Environmental Variability in Caenorhabditis elegans.

Neuron 2015 Apr 9;86(2):428-41. Epub 2015 Apr 9.

Neurosciences Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA; Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA. Electronic address:

The ability to evaluate variability in the environment is vital for making optimal behavioral decisions. Here we show that Caenorhabditis elegans evaluates variability in its food environment and modifies its future behavior accordingly. We derive a behavioral model that reveals a critical period over which information about the food environment is acquired and predicts future search behavior. We also identify a pair of high-threshold sensory neurons that encode variability in food concentration and the downstream dopamine-dependent circuit that generates appropriate search behavior upon removal from food. Further, we show that CREB is required in a subset of interneurons and determines the timescale over which the variability is integrated. Interestingly, the variability circuit is a subset of a larger circuit driving search behavior, showing that learning directly modifies the very same neurons driving behavior. Our study reveals how a neural circuit decodes environmental variability to generate contextually appropriate decisions.
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http://dx.doi.org/10.1016/j.neuron.2015.03.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4409562PMC
April 2015

Light Sheet Fluorescence Microscopy (LSFM).

Curr Protoc Cytom 2015 Jan 5;71:12.37.1-12.37.15. Epub 2015 Jan 5.

Waitt Advanced Biophotonics Center, Salk Institute for Biological Studies, La Jolla, California.

The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane. These approaches, via the placement of a pinhole in the conjugate image plane, provided superior resolution in the axial (z) dimension resulting in nearly isotropic optical sections. However, increased axial resolution, via pinhole optics, comes at the cost of both speed and excitation efficiency. Light sheet fluorescent microscopy (LSFM), a century-old idea made possible with modern developments in both excitation and detection optics, provides sub-cellular resolution and optical sectioning capabilities without compromising speed or excitation efficiency. Over the past decade, several variations of LSFM have been implemented each with its own benefits and deficiencies. Here we discuss LSFM fundamentals and outline the basic principles of several major light-sheet-based imaging modalities (SPIM, inverted SPIM, multi-view SPIM, Bessel beam SPIM, and stimulated emission depletion SPIM) while considering their biological relevance in terms of intrusiveness, temporal resolution, and sample requirements.
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http://dx.doi.org/10.1002/0471142956.cy1237s71DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4294425PMC
January 2015

Characterization of a Mycobacterium tuberculosis nanocompartment and its potential cargo proteins.

J Biol Chem 2014 Jun 22;289(26):18279-89. Epub 2014 May 22.

From the Departments of Molecular Biology and Biochemistry and Pharmaceutical Sciences, University of California, Irvine, California 92697,

Mycobacterium tuberculosis has evolved various mechanisms by which the bacterium can maintain homeostasis under numerous environmental assaults generated by the host immune response. M. tuberculosis harbors enzymes involved in the oxidative stress response that aid in survival during the production of reactive oxygen species in activated macrophages. Previous studies have shown that a dye-decolorizing peroxidase (DyP) is encapsulated by a bacterial nanocompartment, encapsulin (Enc), whereby packaged DyP interacts with Enc via a unique C-terminal extension. M. tuberculosis also harbors an encapsulin homolog (CFP-29, Mt-Enc), within an operon with M. tuberculosis DyP (Mt-DyP), which contains a C-terminal extension. Together these observations suggest that Mt-DyP interacts with Mt-Enc. Furthermore, it has been suggested that DyPs may function as either a heme-dependent peroxidase or a deferrochelatase. Like Mt-DyP, M. tuberculosis iron storage ferritin protein, Mt-BfrB, and an M. tuberculosis protein involved in folate biosynthesis, 7,8-dihydroneopterin aldolase (Mt-FolB), have C-terminal tails that could also interact with Mt-Enc. For the first time, we show by co-purification and electron microscopy that mycobacteria via Mt-Enc can encapsulate Mt-DyP, Mt-BfrB, and Mt-FolB. Functional studies of free or encapsulated proteins demonstrate that they retain their enzymatic activity within the Mt-Enc nanocompartment. Mt-DyP, Mt-FolB, and Mt-BfrB all have antioxidant properties, suggesting that if these proteins are encapsulated by Mt-Enc, then this nanocage may play a role in the M. tuberculosis oxidative stress response. This report provides initial structural and biochemical clues regarding the molecular mechanisms that utilize compartmentalization by which the mycobacterial cell may aid in detoxification of the local environment to ensure long term survival.
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http://dx.doi.org/10.1074/jbc.M114.570119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140288PMC
June 2014

From "There's Plenty of Room at the Bottom" to seeing what is actually there.

Chemphyschem 2014 Mar 3;15(4):547-9. Epub 2014 Mar 3.

Waitt Advanced Biophotonics Center, Salk Institute for Biological Studies, La Jolla, CA (USA).

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http://dx.doi.org/10.1002/cphc.201400097DOI Listing
March 2014

Helium Ion Microscopy (HIM) for the imaging of biological samples at sub-nanometer resolution.

Sci Rep 2013 Dec 17;3:3514. Epub 2013 Dec 17.

Waitt Advanced Biophotonics Center, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.

Scanning Electron Microscopy (SEM) has long been the standard in imaging the sub-micrometer surface ultrastructure of both hard and soft materials. In the case of biological samples, it has provided great insights into their physical architecture. However, three of the fundamental challenges in the SEM imaging of soft materials are that of limited imaging resolution at high magnification, charging caused by the insulating properties of most biological samples and the loss of subtle surface features by heavy metal coating. These challenges have recently been overcome with the development of the Helium Ion Microscope (HIM), which boasts advances in charge reduction, minimized sample damage, high surface contrast without the need for metal coating, increased depth of field, and 5 angstrom imaging resolution. We demonstrate the advantages of HIM for imaging biological surfaces as well as compare and contrast the effects of sample preparation techniques and their consequences on sub-nanometer ultrastructure.
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http://dx.doi.org/10.1038/srep03514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3865489PMC
December 2013