Nanowire facilitated transfer of sensitive TEM samples in a FIB.

We introduce a facile approach to transfer thin films and other mechanically sensitive TEM samples inside a FIB with minimal introduction of stress and bending. The method is making use of a pre-synthetized flexible freestanding Ag nanowire attached to the tip of a typical tungsten micromanipulator inside the FIB. The main advantages of this approach are the significantly reduced stress-induced bending during transfer and attachment of the TEM sample, the very short time required to attach and cut the nanowire, the operation at very low dose and ion current, and only using the e-beam for Pt deposition during the transfer of sensitive TEM samples. This results in a reduced sample preparation time and reduced exposure to the ion beam or e-beam for Pt deposition during the sample preparation and thus also reduced contamination and beam damage. The method was applied to a number of thin films and different TEM samples in order to illustrate the advantageous benefits of the concept. In particular, the technique has been successfully tested for the transfer of a thin film onto a MEMS heating chip for in situ TEM experiments.

Profiling Protease Specificity: Combining Yeast ER Sequestration Screening (YESS) with Next Generation Sequencing.

An enzyme engineering technology involving yeast endoplasmic reticulum (ER) sequestration screening (YESS) has been recently developed. Here, a new method is established, in which the YESS platform is combined with NextGen sequencing (NGS) to enable a comprehensive survey of protease specificity. In this approach, a combinatorial substrate library is targeted to the yeast ER and transported through the secretory pathway, interacting with any protease(s) residing in the ER. Multicolor FACS screening is used to isolate cells labeled with fluorophore-conjugated antibodies, followed by NGS to profile the cleaved substrates. The YESS-NGS method was successfully applied to profile the sequence specificity of the wild-type and an engineered variant of the tobacco etch mosaic virus protease. Proteolysis in the yeast secretory pathway was also mapped for the first time in vivo revealing a major cleavage pattern of Ali/Leu-X-Lys/Arg-Arg. Here Ali is any small aliphatic residue, but especially Leu. This pattern was verified to be due to the well-known endogenous protease Kex2 after comparison to a newly generated Kex2 knockout strain as well as cleavage of peptides with recombinant Kex2 in vitro. This information is particularly important for those using yeast display technology, as library members with Ali/Leu-X-Lys/Arg-Arg patterns are likely being removed from screens via Kex2 cleavage without the researcher's knowledge.

Antibodies and their multivalent constructs for cancer therapy.

In 2008 cancer was identified by the World Health Organization (WHO) as one of four threats to human health and development. Since the early published reports of the first chemotherapeutic, mustine, in 1946, the anti-cancer drug and development industry has grown into a multi-billion dollar business enterprise. Worldwide, the rates of new cancer cases and deaths has been steadily increasing each year, with the estimation by the WHO-sponsored GLOBOCAN cancer database, that at current rates, nearly 13 million cancer deaths will be reported in 2030. The recent successes of monoclonal antibodies (mAbs), an important class of glycoprotein, and their multivalent and drug conjugated derivatives over the past 30 years have led to the approval of 12 monoclonal antibodies for use in cancer treatment by the FDA. Modern recombinant and engineering techniques have led to an explosion of antibody platforms that can be attributed to great gains in clinical efficacy. This review discusses and outlines a sample of mAbs currently approved for cancer treatment by the FDA, as well as antibody platforms in the research pipeline and clinic that have been engineered for greater tumor penetration, binding, and efficacy.

Near-field effects and energy transfer in hybrid metal-oxide nanostructures.

One of the big challenges of the 21st century is the utilization of nanotechnology for energy technology. Nanoscale structures may provide novel functionality, which has been demonstrated most convincingly by successful applications such as dye-sensitized solar cells introduced by M. Grätzel. Applications in energy technology are based on the transfer and conversion of energy. Following the example of photosynthesis, this requires a combination of light harvesting, transfer of energy to a reaction center, and conversion to other forms of energy by charge separation and transfer. This may be achieved by utilizing hybrid nanostructures, which combine metallic and nonmetallic components. Metallic nanostructures can interact strongly with light. Plasmonic excitations of such structures can cause local enhancement of the electrical field, which has been utilized in spectroscopy for many years. On the other hand, the excited states in metallic structures decay over very short lifetimes. Longer lifetimes of excited states occur in nonmetallic nanostructures, which makes them attractive for further energy transfer before recombination or relaxation sets in. Therefore, the combination of metallic nanostructures with nonmetallic materials is of great interest. We report investigations of hybrid nanostructured model systems that consist of a combination of metallic nanoantennas (fabricated by nanosphere lithography, NSL) and oxide nanoparticles. The oxide particles were doped with rare-earth (RE) ions, which show a large shift between absorption and emission wavelengths, allowing us to investigate the energy-transfer processes in detail. The main focus is on TiO2 nanoparticles doped with Eu(3+), since the material is interesting for applications such as the generation of hydrogen by photocatalytic splitting of water molecules. We use high-resolution techniques such as confocal fluorescence microscopy for the investigation of energy-transfer processes. The experiments are supported by simulations of the electromagnetic field enhancement in the vicinity of well-defined nanoantennas. The results show that the presence of the nanoparticle layer can modify the field enhancement significantly. In addition, we find that the fluorescent intensities observed in the experiments are affected by agglomeration of the nanoparticles. In order to further elucidate the possible influence of agglomeration and quenching effects in the vicinity of the nanoantennas, we have used a commercial organic pigment containing Eu, which exhibits an extremely narrow particle size distribution and no significant agglomeration. We demonstrate that quenching of the Eu fluorescence can be suppressed by covering the nanoantennas with a 10 nm thick SiO x layer.

Islet amyloid: from fundamental biophysics to mechanisms of cytotoxicity.

Pancreatic islet amyloid is a characteristic feature of type 2 diabetes. The major protein component of islet amyloid is the polypeptide hormone known as islet amyloid polypeptide (IAPP, or amylin). IAPP is stored with insulin in the ß-cell secretory granules and is released in response to the stimuli that lead to insulin secretion. IAPP is normally soluble and is natively unfolded in its monomeric state, but forms islet amyloid in type 2 diabetes. Islet amyloid is not the cause of type 2 diabetes, but it leads to ß-cell dysfunction and cell death, and contributes to the failure of islet cell transplantation. The mechanism of IAPP amyloid formation is not understood and the mechanisms of cytotoxicity are not fully defined.

Commercial proteases: present and future.

This review presents a brief overview of the general categories of commercially used proteases, and critically surveys the successful strategies currently being used to improve the properties of proteases for various commercial purposes. We describe the broad application of proteases in laundry detergents, food processing, and the leather industry. The review also introduces the expanding development of proteases as a class of therapeutic agents, as well as highlighting recent progress in the field of protease engineering. The potential commercial applications of proteases are rapidly growing as recent technological advances are producing proteases with novel properties and substrate specificities.

Ionic strength effects on amyloid formation by amylin are a complicated interplay among Debye screening, ion selectivity, and Hofmeister effects.

Amyloid formation plays a role in a wide range of human diseases. The rate and extent of amyloid formation depend on solution conditions, including pH and ionic strength. Amyloid fibrils often adopt structures with parallel, in-register ß-sheets, which generate quasi-infinite arrays of aligned side chains. These arrangements can lead to significant electrostatic interactions between adjacent polypeptide chains. The effect of ionic strength and ion composition on the kinetics of amyloid formation by islet amyloid polypeptide (IAPP) is examined. IAPP is a basic 37-residue polypeptide responsible for islet amyloid formation in type 2 diabetes. Poisson-Boltzmann calculations revealed significant electrostatic repulsion in a model of the IAPP fibrillar state. The kinetics of IAPP amyloid formation are strongly dependent on ionic strength, varying by a factor of >10 over the range of 20-600 mM NaCl at pH 8.0, but the effect is not entirely due to Debye screening. At low ionic strengths, the rate depends strongly on the identity of the anion, varying by a factor of nearly 4, and scales with the electroselectivity series, implicating anion binding. At high ionic strengths, the rate varies by only 8% and scales with the Hofmeister series. At intermediate ionic strengths, no clear trend is detected, likely because of the convolution of different effects. The effects of salts on the growth phase and lag phase of IAPP amyloid formation are strongly correlated. At pH 5.5, where the net charge on IAPP is higher, the effect of different anions scales with the electroselectivity series at all salt concentrations.

Deamidation accelerates amyloid formation and alters amylin fiber structure.

Deamidation of asparagine and glutamine is the most common nonenzymatic, post-translational modification. Deamidation can influence the structure, stability, folding, and aggregation of proteins and has been proposed to play a role in amyloid formation. However there are no structural studies of the consequences of deamidation on amyloid fibers, in large part because of the difficulty of studying these materials using conventional methods. Here we examine the effects of deamidation on the kinetics of amyloid formation by amylin, the causative agent of type 2 diabetes. We find that deamidation accelerates amyloid formation and the deamidated material is able to seed amyloid formation by unmodified amylin. Using site-specific isotope labeling and two-dimensional infrared (2D IR) spectroscopy, we show that fibers formed by samples that contain deamidated polypeptide contain reduced amounts of ß-sheet. Deamidation leads to disruption of the N-terminal ß-sheet between Ala-8 and Ala-13, but ß-sheet is still retained near Leu-16. The C-terminal sheet is disrupted near Leu-27. Analysis of potential sites of deamidation together with structural models of amylin fibers reveals that deamidation in the N-terminal ß-sheet region may be the cause for the disruption of the fiber structure at both the N- and C-terminal ß-sheet. Thus, deamidation is a post-translational modification that creates fibers that have an altered structure but can still act as a template for amylin aggregation. Deamidation is very difficult to detect with standard methods used to follow amyloid formation, but isotope-labeled IR spectroscopy provides a means for monitoring sample degradation and investigating the structural consequences of deamidation.

Two-dimensional infrared spectroscopy reveals the complex behaviour of an amyloid fibril inhibitor.

Amyloid formation has been implicated in the pathology of over 20 human diseases, but the rational design of amyloid inhibitors is hampered by a lack of structural information about amyloid-inhibitor complexes. We use isotope labelling and two-dimensional infrared spectroscopy to obtain a residue-specific structure for the complex of human amylin (the peptide responsible for islet amyloid formation in type 2 diabetes) with a known inhibitor (rat amylin). Based on its sequence, rat amylin should block formation of the C-terminal ß-sheet, but at 8 h after mixing, rat amylin blocks the N-terminal ß-sheet instead. At 24 h after mixing, rat amylin blocks neither ß-sheet and forms its own ß-sheet, most probably on the outside of the human fibrils. This is striking, because rat amylin is natively disordered and not previously known to form amyloid ß-sheets. The results show that even seemingly intuitive inhibitors may function by unforeseen and complex structural processes.

Anisotropic organization and microscopic manipulation of self-assembling synthetic porphyrin microrods that mimic chlorosomes: bacterial light-harvesting systems.

Being able to control in time and space the positioning, orientation, movement, and sense of rotation of nano- to microscale objects is currently an active research area in nanoscience, having diverse nanotechnological applications. In this paper, we demonstrate unprecedented control and maneuvering of rod-shaped or tubular nanostructures with high aspect ratios which are formed by self-assembling synthetic porphyrins. The self-assembly algorithm, encoded by appended chemical-recognition groups on the periphery of these porphyrins, is the same as the one operating for chlorosomal bacteriochlorophylls (BChl's). Chlorosomes, rod-shaped organelles with relatively long-range molecular order, are the most efficient naturally occurring light-harvesting systems. They are used by green photosynthetic bacteria to trap visible and infrared light of minute intensities even at great depths, e.g., 100 m below water surface or in volcanic vents in the absence of solar radiation. In contrast to most other natural light-harvesting systems, the chlorosomal antennae are devoid of a protein scaffold to orient the BChl's; thus, they are an attractive goal for mimicry by synthetic chemists, who are able to engineer more robust chromophores to self-assemble. Functional devices with environmentally friendly chromophores-which should be able to act as photosensitizers within hybrid solar cells, leading to high photon-to-current conversion efficiencies even under low illumination conditions-have yet to be fabricated. The orderly manner in which the BChl's and their synthetic counterparts self-assemble imparts strong diamagnetic and optical anisotropies and flow/shear characteristics to their nanostructured assemblies, allowing them to be manipulated by electrical, magnetic, or tribomechanical forces.

2DIR spectroscopy of human amylin fibrils reflects stable ß-sheet structure.

The aggregation of human amylin to form amyloid contributes to islet ß-cell dysfunction in type 2 diabetes. Studies of amyloid formation have been hindered by the low structural resolution or relatively modest time resolution of standard methods. Two-dimensional infrared (2DIR) spectroscopy, with its sensitivity to protein secondary structures and its intrinsic fast time resolution, is capable of capturing structural changes during the aggregation process. Moreover, isotope labeling enables the measurement of residue-specific information. The diagonal line widths of 2DIR spectra contain information about dynamics and structural heterogeneity of the system. We illustrate the power of a combined atomistic molecular dynamics simulation and theoretical and experimental 2DIR approach by analyzing the variation in diagonal line widths of individual amide I modes in a series of labeled samples of amylin amyloid fibrils. The theoretical and experimental 2DIR line widths suggest a "W" pattern, as a function of residue number. We show that large line widths result from substantial structural disorder and that this pattern is indicative of the stable secondary structure of the two ß-sheet regions. This work provides a protocol for bridging MD simulation and 2DIR experiments for future aggregation studies.

Efficient microwave-assisted synthesis of human islet amyloid polypeptide designed to facilitate the specific incorporation of labeled amino acids.

A cost-efficient, time-reducing solid-phase synthesis of the amyloidogenic, 37 residue islet amyloid polypeptide (IAPP) is developed using two pseudoprolines (highlighted blue in sequence) in combination with microwave technology. A yield twice that obtained with conventional syntheses is realized. The utility of this protocol is demonstrated by the synthesis of a (13)C(18)O-labeled Ser-20 IAPP variant, a prohibitively expensive and chemically challenging site to label via other protocols. TEM analysis shows the peptide forms normal amyloid (abstract image).

Residue-specific, real-time characterization of lag-phase species and fibril growth during amyloid formation: a combined fluorescence and IR study of p-cyanophenylalanine analogs of islet amyloid polypeptide.

Amyloid formation normally exhibits a lag phase followed by a growth phase, which leads to amyloid fibrils. Characterization of the species populated during the lag phase is experimentally challenging, but is critical since the most toxic entities may be pre-fibrillar species. p-Cyanophenylalanine (F(C[triple bond]N)) fluorescence is used to probe the nature of lag-phase species populated during the formation of amyloid by human islet amyloid polypeptide. The polypeptide contains two phenylalanines at positions 15 and 23 and a single tyrosine located at the C-terminus. Each aromatic residue was separately replaced by F(C[triple bond]N). The substitutions do not perturb amyloid formation relative to wild-type islet amyloid polypeptide as detected using thioflavin T fluorescence and electron microscopy. F(C[triple bond]N) fluorescence is high when the cyano group is hydrogen bonded and low when it is not. It can also be quenched via Förster resonance energy transfer to tyrosine. Fluorescence intensity was monitored in real time and revealed that all three positions remained exposed to solvent during the lag phase but less exposed than unstructured model peptides. The time course of amyloid formation as monitored by thioflavin T fluorescence and F(C[triple bond]N) fluorescence is virtually identical. Fluorescence quenching experiments confirmed that each residue remains exposed during the lag phase. These results place significant constraints on the nature of intermediates that are populated during the lag phase and indicate that significant sequestering of the aromatic side chains does not occur until beta-structure sufficient to bind thioflavin T has developed. Seeding studies and analysis of maximum rates confirm that sequestering of the cyano groups occurs concomitantly with the development of thioflavin T binding capability. Overall, the process of amyloid formation and growth appears to be remarkably homogenous in terms of side-chain ordering. F(C[triple bond]N) also provides information about fibril structure. Fluorescence emission measurements, infrared measurements, and quenching studies indicate that the aromatic residues are differentially exposed in the fibril state with Phe15 being the most exposed. F(C[triple bond]N) is readily accommodated into proteins; thus, the approach should be broadly applicable.

Modulation of p-cyanophenylalanine fluorescence by amino acid side chains and rational design of fluorescence probes of alpha-helix formation.

p-Cyanophenylalanine is an extremely useful fluorescence probe of protein structure that can be recombinantly and chemically incorporated into proteins. The probe has been used to study protein folding, protein-membrane interactions, protein-peptide interactions, and amyloid formation; however, the factors that control its fluorescence are not fully understood. Hydrogen bonding to the cyano group is known to play a major role in modulating the fluorescence quantum yield, but the role of potential side-chain quenchers has not yet been elucidated. A systematic study of the effects of different side chains on p-cyanophenylalanine fluorescence is reported. Tyr is found to have the largest effect followed by deprotonated His, Met, Cys, protonated His, Asn, Arg, and protonated Lys. Deprotonated amino groups are much more effective fluorescence quenchers than protonated amino groups. Free neutral imidazole and hydroxide ion are also effective quenchers of p-cyanophenylalanine fluorescence with Stern-Volmer constants of 39.8 and 22.1 M(-1), respectively. The quenching of p-cyanophenylalanine fluorescence by specific side chains is exploited in developing specific, high-sensitivity, fluorescence probes of helix formation. The approach is demonstrated with Ala-based peptides that contain a p-cyanophenylalanine-His or a p-cyanophenylalanine-Tyr pair located at positions i and i + 4. The p-cyanophenylalanine-His pair is most useful when the His side chain is deprotonated and is, thus, complementary to the Trp-His pair which is most sensitive when the His side chain is protonated.

Neprilysin impedes islet amyloid formation by inhibition of fibril formation rather than peptide degradation.

Deposition of islet amyloid polypeptide (IAPP) as islet amyloid in type 2 diabetes contributes to loss of beta-cell function and mass, yet the mechanism for its occurrence is unclear. Neprilysin is a metallopeptidase known to degrade amyloid in Alzheimer disease. We previously demonstrated neprilysin to be present in pancreatic islets and now sought to determine whether it plays a role in degrading islet amyloid. We used an in vitro model where cultured human IAPP (hIAPP) transgenic mouse islets develop amyloid and thereby have increased beta-cell apoptosis. Islet neprilysin activity was inhibited or up-regulated using a specific inhibitor or adenovirus encoding neprilysin, respectively. Following neprilysin inhibition, islet amyloid deposition and beta-cell apoptosis increased by 54 and 75%, respectively, whereas when neprilysin was up-regulated islet amyloid deposition and beta-cell apoptosis both decreased by 79%. To determine if neprilysin modulated amyloid deposition by cleaving hIAPP, analysis of hIAPP incubated with neprilysin was performed by mass spectrometry, which failed to demonstrate neprilysin-induced cleavage. Rather, neprilysin may act by reducing hIAPP fibrillogenesis, which we showed to be the case by fluorescence-based thioflavin T binding studies and electron microscopy. In summary, neprilysin decreases islet amyloid deposition by inhibiting hIAPP fibril formation, rather than degrading hIAPP. These findings suggest that targeting the role of neprilysin in IAPP fibril assembly, in addition to IAPP cleavage by other peptidases, may provide a novel approach to reduce and/or prevent islet amyloid deposition in type 2 diabetes.

Self-assembled chromophores within mesoporous nanocrystalline TiO2: towards biomimetic solar cells.

Artificial light-harvesting antennas consisting of self-assembled chromophores that mimic the natural pigments of photosynthetic bacteria have been inserted into voids induced in porous titania (TiO2, anatase) in order to investigate their suitability for hybrid solar cells. Mesoporous nanocrystalline TiO2 with additional uniform macropores was treated with precursor solutions of the pigment which was then induced to self-assemble within the voids. The chromophores were tailored to combine the self-assembly characteristics of the natural bacteriochlorophylls with the robustness of artificial Zn-porphyrins being stable for prolonged periods even upon heating to over 200 degrees C. They assemble on the TiO2 surface to form nano- to micro-crystalline structures with lengths from tens of nm up to several microm and show a photosensitization effect which is supposed to be dependent on the assembly size. The natural examples of these antennas are found in green sulfur bacteria which are able to use photosynthesis in deep water regions with minute light intensities. The implementation of biomimetic antennas for light harvesting and a better photon management may lead to a rise in efficiency of dye-sensitized solar cells also under low light illumination conditions.

Rifampicin does not prevent amyloid fibril formation by human islet amyloid polypeptide but does inhibit fibril thioflavin-T interactions: implications for mechanistic studies of beta-cell death.

Amyloid formation has been implicated in more than 20 different human diseases, including Alzheimer's disease, Parkinson's disease, and type 2 diabetes. The development of inhibitors of amyloid is a topic of considerable interest, both because of their potential therapeutic applications and because they are useful mechanistic probes. Recent studies have highlighted the potential use of rifampicin as an inhibitor of amyloid formation by a variety of polypeptides; however, there are conflicting reports on its ability to inhibit amyloid formation by islet amyloid polypeptide (IAPP). IAPP is the cause of islet amyloid in type 2 diabetes. We show that rifampicin does not prevent amyloid formation by IAPP and does not disaggregate preformed IAPP amyloid fibrils;, instead, it interferes with standard fluorescence-based assays of amyloid formation. Rifampicin is unstable in aqueous solution and is readily oxidized. However, the effects of oxidized and reduced rifampicin are similar, in that neither prevents amyloid formation by IAPP. Furthermore, use of a novel p-cyanoPhe analogue of IAPP shows that rifampicin does not significantly affect the kinetics of IAPP amyloid formation. The implications for the development of amyloid inhibitors are discussed as are the implications for studies of the toxicity of islet amyloid. The work also demonstrates the utility of p-cyanoPhe IAPP for the screening of inhibitors. The data indicate that rifampicin cannot be used to test the relative toxicity of IAPP fibrils and prefibril aggregates of IAPP.

Aromatic interactions are not required for amyloid fibril formation by islet amyloid polypeptide but do influence the rate of fibril formation and fibril morphology.

Amyloid formation has been implicated in a wide range of human diseases, and a diverse set of proteins is involved. There is considerable interest in elucidating the interactions which lead to amyloid formation and which contribute to amyloid fibril stability. Recent attention has been focused upon the potential role of aromatic-aromatic and aromatic-hydrophobic interactions in amyloid formation by short to midsized polypeptides. Here we examine whether aromatic residues are necessary for amyloid formation by islet amyloid polypeptide (IAPP). IAPP is responsible for the formation of islet amyloid in type II diabetes which is thought to play a role in the pathology of the disease. IAPP is 37 residues in length and contains three aromatic residues, Phe-15, Phe-23, and Tyr-37. Structural models of IAPP amyloid fibrils postulate that Tyr-37 is near one of the phenylalanine residues, and it is known that Tyr-37 interacts with one of the phenylalanines during fibrillization; however, it is not known if aromatic-aromatic or aromatic-hydrophobic interactions are absolutely required for amyloid formation. An F15L/F23L/Y37L triple mutant (IAPP-3XL) was prepared, and its ability to form amyloid was tested. CD, thioflavin binding assays, AFM, and TEM measurements all show that the triple leucine mutant readily forms amyloid fibrils. The substitutions do, however, decrease the rate of fibril formation and alter the tendency of fibrils to aggregate. Thus, while aromatic residues are not an absolute requirement for amyloid formation by IAPP, they do play a role in the fibril assembly process.