Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.
21 Related JoVE Articles!
Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides
Institutions: The Hebrew University of Jerusalem.
In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro
will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2
. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8
. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a "beads on a string"-like structure by this process.9
The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.
Chemistry, Issue 81, Materials (General), self-assembly, peptides, diphenylalanine, atomatic interactions, coassembly, molecular recognition
Bacterial Immobilization for Imaging by Atomic Force Microscopy
Institutions: Oak Ridge National Laboratory, University of Tennessee , Eastern Virginia Medical School, Oak Ridge National Laboratory.
AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.
Bioengineering, Issue 54, Bacteria, AFM imaging, Liquid imaging, Gelatin, Bacterial Immobilization
Assaying β-amyloid Toxicity using a Transgenic C. elegans Model
Institutions: University of Colorado, University of Colorado.
Accumulation of the β-amyloid peptide (Aβ) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. Aβ is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans
can be transgenically engineered to express human Aβ. Depending on the tissue or timing of Aβ expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of Aβ toxicity. For example, transgenic worms with pan-neuronal Aβ expression have defects is associative learning (Dosanjh et al.
2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al.
2006). One particularly useful C. elegans
model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an Aβ transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al.
2003). Treatments that counter Aβ toxicity in this model [e.g., expression of a protective transgene (Hassan et al.
2009) or exposure to Ginkgo biloba extracts (Wu et al.
2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans
model, with particular attention to experimental variables that can influence this measurement.
Neuroscience, Issue 44, Alzheimer's disease, paralysis, compound screening, Inclusion Body Myositis, invertebrate model
Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides
Institutions: University of California, Los Angeles, University of California, Los Angeles, University of California, Los Angeles.
The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization1
. Mechanistically, PICUP involves photo-oxidation of Ru2+
in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru3+
by irradiation with visible light in the presence of an electron acceptor. Ru3+
is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical1,2
. Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods3,4
include short (≤1 s) exposure to non-destructive visible light, no need for pre facto
modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid β-protein variants (Aβ40 and Aβ42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF).
Cross-linking, Issue 23, PICUP, amyloid β-protein, oligomer, amyloid, protein assembly
Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model
Institutions: The University of British Columbia.
Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid β protein (Aβ)1
, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by β-secretase and γ-secretase. The amyloid hypothesis entails that Aγ-containing plaques as the underlying toxic mechanism in AD pathology2
. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of Aγ neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.
Neuroscience, Issue 53, Alzheimer’s disease, neuritic plaques, Amyloid β protein, APP, transgenic mouse
Selection of Aptamers for Amyloid β-Protein, the Causative Agent of Alzheimer's Disease
Institutions: David Geffen School of Medicine, University of California, Los Angeles, University of California, Los Angeles.
Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1
. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3
Amyloid β-protein (Aβ) is central to AD pathogenesis. Soluble, oligomeric Aβ assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5
. Various forms of soluble Aβ assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6
. Specific molecular recognition tools may unravel the relationships amongst Aβ assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8
. Aptamers are oligonucleotides generated by in-vitro
selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10
. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11
, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16
. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-β17
, a soluble, oligomeric, β-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18
. Aptamers generated using monomeric and several forms of fibrillar β2
m) were found to bind fibrils of certain other amyloidogenic proteins besides β2
. Ylera et al
. described RNA aptamers selected against immobilized monomeric Aβ4020
. Unexpectedly, these aptamers bound fibrillar Aβ40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric Aβ4021
generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23
. Similar to previous findings17,19,20
, these aptamers reacted with fibrils of Aβ and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21
. Here, we present the SELEX methodology used in production of these aptamers21
Neuroscience, Issue 39, Cellular Biology, Aptamer, RNA, amyloid β-protein, oligomer, amyloid fibrils, protein assembly
In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen
Institutions: University of Toronto, University of Toronto.
Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo
through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro
, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.
Bioengineering, Issue 67, Chemistry, Biochemistry, Tissue Engineering, Collagen, Self-assembly, Native, Fibrous long spacing, Segmental long spacing, AFM, atomic force microscopy
Purification of Hsp104, a Protein Disaggregase
Institutions: University of Pennsylvania.
Hsp104 is a hexameric AAA+ protein1
from yeast, which couples ATP hydrolysis to protein disaggregation2-10
(Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12
. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22
. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30
. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli
orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35
. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38
. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23
as well as amyloid forms of PrP39
. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23
and Huntington's disease38
. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7
. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46
. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli
. The use of E. coli
allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6
-tag removal compared to a previous purification method from E. coli47
. Moreover, our protocol is more facile and convenient than two more recent protocols26,48
Molecular Biology, Issue 55, Neuroscience, Hsp104, AAA+, disaggregase, heat shock, amyloid, prion
Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro
Institutions: University of Central Florida.
The development of more predictive and biologically relevant in vitro
assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro,
leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.
Bioengineering, Issue 92, cantilever, in vitro, contraction, skeletal muscle, NMJ, cardiomyocytes, functional
Preparation of Oligomeric β-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices
Institutions: Columbia University.
Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). β-amyloid (Aβ), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by Aβ is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that Aβ produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic Aβ1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric Aβ1-42
. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized Aβ1-42
compared to slices in a control experiment where no Aβ1-42
exposure had occurred.
JoVE Neuroscience, Issue 41, brain, mouse, hippocampus, plasticity, LTP, amyloid
Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy
Institutions: University of Sydney, Dankook University.
Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3
Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.
Chemistry, Issue 89,
Colloidal Probe, Nanoscopy, Suspension Stability, Adhesion Mapping, Force, Particle Interaction, Particle Kinetics
Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy
Institutions: University of Guelph.
Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus
USA100 cells on stainless steel and gold using KPFM.
Bioengineering, Issue 93, Kelvin probe force microscopy, atomic force microscopy, surface potential, stainless steel, microbial attachment, bacterial biofilms, methicillin-resistant Staphylococcus aureus
Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation
Institutions: Virginia Tech, Virginia Tech, Illinois Institute of Technology- Moffett Campus, University of Guadalajara, Virginia Tech, Virginia Tech.
Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.
Plant Biology, Issue 88, nanocellulose, thin films, quartz crystal microbalance, layer-by-layer, LbL
In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions
Institutions: Pacific Northwest National Laboratory.
Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ
characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3
(bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ
time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ
Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ
IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.
Chemistry, Issue 88, soft landing, mass selected ions, electrospray, secondary ion mass spectrometry, infrared spectroscopy, organometallic, catalysis
Performing and Processing FNA of Anterior Fat Pad for Amyloid
Institutions: Medical College of Wisconsin, Wayne State University School of Medicine Detroit Medical Center, Medical College of Wisconsin, Medical College of Wisconsin, Medical College of Wisconsin.
Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis. Since the clinical presentation of this disease is so variable and non-specific, the associated risks of these biopsies are too great for the diagnostic yield. Other sites that have a lower biopsy risk, such as skin or gingival, are also relatively invasive and expensive. In addition, these biopsies may not always have sufficient amyloid deposits to establish a diagnosis. Fat pad aspiration has demonstrated good clinical correlation with low cost and minimal morbidity. However, there are no standardized protocols for performing this procedure or processing the aspirated specimen, which leads to variable and nonreproducible results. The most frequently utilized modality for detecting amyloid in tissue is an apple-green birefringence on Congo red stained sections using a polarizing microscope. This technique requires cell block preparation of aspirated material. Unfortunately, patients presenting in early stage of amyloidosis have minimal amounts of amyloid which greatly reduces the sensitivity of Congo red stained cell block sections of fat pad aspirates. Therefore, ultrastructural evaluation of fat pad aspirates by electron microscopy should be utilized, given its increased sensitivity for amyloid detection. This article demonstrates a simple and reproducible procedure for performing anterior fat pad aspiration for the detection of amyloid utilizing both Congo red staining of cell block sections and electron microscopy for ultrastructural identification.
Medicine, Issue 44, AL amyloidosis, Congo Red, abdominal fat pad biopsy, electron microscopy, ultrastructural evaluation
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (https://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Characterization of Surface Modifications by White Light Interferometry: Applications in Ion Sputtering, Laser Ablation, and Tribology Experiments
Institutions: Argonne National Laboratory, Argonne National Laboratory, MassThink LLC.
In materials science and engineering it is often necessary to obtain quantitative measurements of surface topography with micrometer lateral resolution. From the measured surface, 3D topographic maps can be subsequently analyzed using a variety of software packages to extract the information that is needed.
In this article we describe how white light interferometry, and optical profilometry (OP) in general, combined with generic surface analysis software, can be used for materials science and engineering tasks. In this article, a number of applications of white light interferometry for investigation of surface modifications in mass spectrometry, and wear phenomena in tribology and lubrication are demonstrated. We characterize the products of the interaction of semiconductors and metals with energetic ions (sputtering), and laser irradiation (ablation), as well as ex situ
measurements of wear of tribological test specimens.
Specifically, we will discuss:
Aspects of traditional ion sputtering-based mass spectrometry such as sputtering rates/yields measurements on Si and Cu and subsequent time-to-depth conversion.
Results of quantitative characterization of the interaction of femtosecond laser irradiation with a semiconductor surface. These results are important for applications such as ablation mass spectrometry, where the quantities of evaporated material can be studied and controlled via pulse duration and energy per pulse. Thus, by determining the crater geometry one can define depth and lateral resolution versus experimental setup conditions.
Measurements of surface roughness parameters in two dimensions, and quantitative measurements of the surface wear that occur as a result of friction and wear tests.
Some inherent drawbacks, possible artifacts, and uncertainty assessments of the white light interferometry approach will be discussed and explained.
Materials Science, Issue 72, Physics, Ion Beams (nuclear interactions), Light Reflection, Optical Properties, Semiconductor Materials, White Light Interferometry, Ion Sputtering, Laser Ablation, Femtosecond Lasers, Depth Profiling, Time-of-flight Mass Spectrometry, Tribology, Wear Analysis, Optical Profilometry, wear, friction, atomic force microscopy, AFM, scanning electron microscopy, SEM, imaging, visualization
Atomically Defined Templates for Epitaxial Growth of Complex Oxide Thin Films
Institutions: University of Twente.
Atomically defined substrate surfaces are prerequisite for the epitaxial growth of complex oxide thin films. In this protocol, two approaches to obtain such surfaces are described. The first approach is the preparation of single terminated perovskite SrTiO3
(001) and DyScO3
(110) substrates. Wet etching was used to selectively remove one of the two possible surface terminations, while an annealing step was used to increase the smoothness of the surface. The resulting single terminated surfaces allow for the heteroepitaxial growth of perovskite oxide thin films with high crystalline quality and well-defined interfaces between substrate and film. In the second approach, seed layers for epitaxial film growth on arbitrary substrates were created by Langmuir-Blodgett (LB) deposition of nanosheets. As model system Ca2
nanosheets were used, prepared by delamination of their layered parent compound HCa2
. A key advantage of creating seed layers with nanosheets is that relatively expensive and size-limited single crystalline substrates can be replaced by virtually any substrate material.
Chemistry, Issue 94, Substrates, oxides, perovskites, epitaxy, thin films, single termination, surface treatment, nanosheets, Langmuir-Blodgett
Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy
Institutions: The Molecular Foundry.
Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2
, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3
. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high‐resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5
. Moreover, OpNS can be a high‐throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6
. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.
Environmental Sciences, Issue 90, small and asymmetric protein structure, electron microscopy, optimized negative staining
Synthesis and Functionalization of Nitrogen-doped Carbon Nanotube Cups with Gold Nanoparticles as Cork Stoppers
Institutions: University of Pittsburgh.
Nitrogen-doped carbon nanotubes consist of many cup-shaped graphitic compartments termed as nitrogen-doped carbon nanotube cups (NCNCs). These as-synthesized graphitic nanocups from chemical vapor deposition (CVD) method were stacked in a head-to-tail fashion held only through noncovalent interactions. Individual NCNCs can be isolated out of their stacking structure through a series of chemical and physical separation processes. First, as-synthesized NCNCs were oxidized in a mixture of strong acids to introduce oxygen-containing defects on the graphitic walls. The oxidized NCNCs were then processed using high-intensity probe-tip sonication which effectively separated the stacked NCNCs into individual graphitic nanocups. Owing to their abundant oxygen and nitrogen surface functionalities, the resulted individual NCNCs are highly hydrophilic and can be effectively functionalized with gold nanoparticles (GNPs), which preferentially fit in the opening of the cups as cork stoppers. These graphitic nanocups corked with GNPs may find promising applications as nanoscale containers and drug carriers.
Physics, Issue 75, Chemistry, Chemical Engineering, Materials Science, Physical Chemistry, Nanotechnology, Metal Nanoparticles, carbon nanotubes (synthesis and properties), carbon nanotubes, chemical vapor deposition, CVD, gold nanoparticles, probe-tip sonication, nitrogen-doped carbon nanotube cups, nanotubes, nanoparticles, nanomaterial, synthesis
Proper Care and Cleaning of the Microscope
Institutions: University of Texas Health Science Center at San Antonio (UTHSCSA).
Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.
Basic Protocols, Issue 18, Current Protocols Wiley, Microscopy, Cleaning the Microscope