Invited commentary.
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Deglutition Disorders↗
Biomedical subjects
Mark Allen
Publications and source records attributed to Mark Allen.
Targeting of cancer cells with ferrimagnetic ferritin cage nanoparticles.
Protein cage architectures such as virus capsids and ferritins are versatile nanoscale platforms amenable to both genetic and chemical modification. Incorporation of multiple functionalities within these nanometer-sized protein architectures demonstrate their potential to serve as functional nanomaterials with applications in medical imaging and therapy. In the present study, we synthesized an iron oxide (magnetite) nanoparticle within the interior cavity of a genetically engineered human H-chain ferritin (HFn). A cell-specific targeting peptide, RGD-4C which binds alphavbeta3 integrins upregulated on tumor vasculature, was genetically incorporated on the exterior surface of HFn. Both magnetite-containing and fluorescently labeled RGD4C-Fn cages bound C32 melanoma cells in vitro. Together these results demonstrate the capability of a genetically modified protein cage architecture to serve as a multifunctional nanoscale container for simultaneous iron oxide loading and cell-specific targeting.
Cell Line, Tumor↗
Dps-like protein from the hyperthermophilic archaeon Pyrococcus furiosus.
Oxidative stress is a universal phenomenon experienced by organisms in all domains of life. Proteins like those in the ferritin-like di-iron carboxylate superfamily have evolved to manage this stress. Here we describe the cloning, isolation, and characterization of a Dps-like protein from the hyperthermophilic archaeon Pyrococcus furiosus (PfDps-like). Phylogenetic analysis, primary structure alignments and higher order structural predictions all suggest that the P. furiosus protein is related to proteins within the broad superfamily of ferritin-like di-iron carboxylate proteins. The recombinant PfDps protein self-assembles into a 12 subunit quaternary structure with an outer shell diameter of approximately 10nm and an interior diameter of approximately 5 nm. Dps proteins functionally manage the toxicity of oxidative stress by sequestering intracellular ferrous iron and using it to reduce H(2)O(2) in a two electron process to form water. The iron is converted to a benign form as Fe(III) within the protein cage. This Dps-mediated reduction of hydrogen peroxide, coupled with the protein's capacity to sequester iron, contributes to its service as a multifunctional antioxidant.
Amino Acid Sequence↗
Structural transitions in Cowpea chlorotic mottle virus (CCMV).
Viral capsids act as molecular containers for the encapsulation of genomic nucleic acid. These protein cages can also be used as constrained reaction vessels for packaging and entrapment of synthetic cargos. The icosahedral Cowpea chlorotic mottle virus (CCMV) is an excellent model for understanding the encapsulation and packaging of both genomic and synthetic materials. High-resolution structural information of the CCMV capsid has been invaluable for evaluating structure-function relationships in the assembled capsid but does not allow insight into the capsid dynamics. The dynamic nature of the CCMV capsid might play an important role in the biological function of the virus. The CCMV capsid undergoes a pH and metal ion dependent reversible structural transition where 60 separate pores in the capsid open or close, exposing the interior of the protein cage to the bulk medium. In addition, the highly basic N-terminal domain of the capsid, which is disordered in the crystal structure, plays a significant role in packaging the viral cargo. Interestingly, in limited proteolysis and mass spectrometry experiments the N-terminal domain is the first part of the subunit to be cleaved, confirming its dynamic nature. Based on our fundamental understanding of the capsid dynamics in CCMV, we have utilized these aspects to direct packaging of a range of synthetic materials including drugs and inorganic nanoparticles.
Bromovirus↗
The structure of the major transition state for folding of an FF domain from experiment and simulation.
We have analysed the transition state of folding of the four-helix FF domain from HYPA/FBP11 by high-resolution experiment and simulation as part of a continuing effort to understand the principles of folding and the refinement of predictive methods. The major transition state for folding was subjected to a Phi-value analysis utilising 50 mutants. The transition state contained a nucleus for folding centred around the end of helix 1 (H1) and the beginning of helix 2 (H2). Secondary structure in this region was fully formed (PhiF=0.9-1) and tertiary interactions were well developed. Interactions in the distal part of the native structure were weak (PhiF=0-0.2). The hydrophobic core and other parts of the protein displayed intermediate Phi-values, suggesting that interactions coalesce as the end of H1 and beginning of H2 are in the process of being formed. The distribution of Phi-values resembled that of barnase, which folds via an intermediate, rather than that of CI2 which folds by a concerted nucleation-condensation mechanism. The overall picture of the transition state structure identified in molecular dynamics simulations is in qualitative agreement, with the turn connecting H1 and H2 being formed, a loosened core, and H4 partially unfolded and detached from the core. There are some differences in the details and interpretation of specific Phi-values.
Carrier Proteins↗
Chip electrospray mass spectrometry for carbohydrate analysis.
Currently two types of chip systems are used in conjunction with MS: out-of-plane devices, where hundreds of nozzles, nanospray emitters are integrated onto a single silicon substrate from which electrospray is established perpendicular to the substrate, and planar microchips, embedding a microchannel at the end of which electrospray is generated in-plane, on the edge of the microchip. In the last two years, carbohydrate research greatly benefited from the introduction and implementation of the chip-based MS. In two laboratories the advantages of the chip electrospray in terms of ionization efficiency, sensitivity, reproducibility, quality of data in combination with high mass accuracy, and resolution of detection were systematically explored for several carbohydrate classes: O- and N-glycopeptides, oligosaccharides, gangliosides and glycoprotein-derived O- and N-glycans, and glycopeptides. The current state-of-the-art in interfacing the chip electrospray devices to high-performance MS for carbohydrate analysis, and the particular requirements for method optimization in both positive and negative ion modes are reviewed here. The recent applications of these miniaturized devices and their general potential for glycomic-based surveys are highlighted.
Amino Acid Sequence↗
Paramagnetic viral nanoparticles as potential high-relaxivity magnetic resonance contrast agents.
In order to compensate for the inherent high threshold of detectability of MR contrast agents, there has been an active interest in the development of paramagnetic nanoparticles incorporating high payloads of Gd(3+) with high molecular relaxivities. Toward this end, the protein cage of Cowpea chlorotic mottle virus (CCMV), having 180 metal binding sites, is being explored. In vivo CCMV binds Ca(2+) at specific metal binding sites; however, Gd(3+) can also bind at these sites. Using fluorescence resonance energy transfer we have characterized the binding affinity of Gd(3+) to the metal binding sites by competition experiments with Tb(3+). The measured dissociation constant (K(d)) for Gd(3+) bound to the virus is 31 microM. The T(1) and T(2) relaxivities of solvent water protons in the presence of Gd(3+)-bound CCMV were 202 and 376 mM(-1) s(-1), respectively, at 61 MHz Larmor frequency. The unusually high relaxivity values of the Gd(3+)-CCMV are largely a result of the nanoparticle virus size and the large number of Gd(3+) ions bound to the virus. These preliminary results should encourage further investigations into the use of viral protein cages as a new platform for MR contrast agents.
Contrast Media↗
Choral reading with filtered speech: effect on stuttering.
This study investigated use of choral reading with filtered components of speech and whispered speech on the frequency of stuttering. Three passages read by a normal adult male were lowpass filtered with kneepoint frequencies at 100 Hz (approximate glottal source), 500 Hz (source and first formant), and 1 kHz (source and the first two formants). Along with a whispered passage, a normal passage, and a control condition, these stimuli were used in a repeated-measures design with 12 adult stutterers as they read passages while listening to one of the stimuli. Frequencies of stuttering in each condition were analyzed. The choral speech, the 500-Hz, the 1-kHz, and the whispered speech conditions all decreased the frequency of stuttering while the 100-Hz stimuli did not. It is suggested that articulatory events, chiefly the encoded speech output from the vocal tract, create effective cues and may induce fluent speech in people who stutter.
Acoustic Stimulation↗
Iron and cobalt oxide and metallic nanoparticles prepared from ferritin.
Metallic Fe and Co and Fe- and Co-based oxide nanoparticles were prepared by a novel method utilizing the biologically relevant protein ferritin. In particular, iron and cobalt oxyhydroxide nanoparticles were assembled within horse spleen and Listeria innocua derived ferritin, respectively, in the aqueous phase. Ferritin containing either Fe or Co oxide was transferred and dried on a SiO2 support where the protein shell was removed during exposure to a highly oxidizing environment. It was also shown that the metal oxide particles could be reduced to the respective metal by heating in hydrogen. X-ray photoelectron spectroscopy was used to characterize the composition of the particles and atomic force microscopy was used to characterize the size of the nanoparticles. Depending on the Fe or Co loading and/or type of ferritin used, metallic and oxide nanoparticles could be produced within a range of 20-60 A.
Animals↗
Laboratory evidence for a key intermediate in the Venus atmosphere: peroxychloroformyl radical.
For two decades, the peroxychloroformyl radical, ClC(O)OO, has played a central role in models of the chemical stability of the Venus atmosphere. No confirmation, however, has been possible in the absence of laboratory measurements for ClC(O)OO. We report the isolation of ClC(O)OO in a cryogenic matrix and its infrared and ultraviolet spectral signatures. These experiments show that ClC(O)OO is thermally and photolytically stable in the Venus atmosphere. These experimental discoveries validate the existence of ClC(O)OO, confirm several longstanding model assumptions, and provide a basis for the astronomical search for this important radical species.
Journal Article↗
Fully automated chip-based mass spectrometry for complex carbohydrate system analysis.
Carbohydrates represent a major class of biopolymers, which occur in nature either as oligosaccharides or glycoconjugates, in which the sugar moiety is linked to proteins or lipids. The significance of mass spectrometry for highly sensitive analysis of complex carbohydrates increased after the introduction of the electrospray ionization and matrix assisted laser desorption/ionization methods and the possibility of tandem MS for sequencing of single molecular species in complex mixtures. Rapid and sensitive characterization of carbohydrates in biological systems by automated nanoscale liquid delivery and chip-based electrospray interface techniques have not been developed so far. In this contribution, the implementation and optimization of a fully automated chip-based nanoelectrospray assembly (NanoMate system), operating in the negative ion mode, in combination with QTOF-tandem MS for mapping/sequencing and computer-assisted structure assignment for carbohydrate components in complex mixtures is presented.
Automation↗
Simultaneous selected reaction monitoring, MS/MS and MS3 quantitation for the analysis of pharmaceutical compounds in human plasma using chip-based infusion.
An assay method with mass spectrometric detection was developed for the quantitative analysis of a pharmaceutical compound and its major metabolite in human plasma using chip-based infusion. Liquid-liquid extraction sample preparation was found to be essential to minimize matrix suppression and to achieve a limit of quantitation (LOQ) of 2.5 ng/mL using a 100 microL plasma aliquot. The potential for simultaneous quantitation in selected reaction monitoring (SRM), tandem mass spectrometry (MS/MS) (enhanced product ion), and MS(3) was investigated and found to be very beneficial in improving assay selectivity. A novel concept for monitoring quantitative assay performance using a SRM/MS(3) ratio is proposed.
Mass Spectrometry↗
Coupling of fully automated chip electrospray to Fourier transform ion cyclotron resonance mass spectrometry for high-performance glycoscreening and sequencing.
The NanoMate robot has been coupled to a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer at 9.4 T and implemented for the first time for complex carbohydrate analysis. It was optimized in the negative ion mode to achieve automated sample delivery on the chip along with increased sensitivity, ultra-high resolution and accurate mass determination. A novel bracket has been designed to allow a reliable mounting of the NanoMate to the Apollo electrospray ionization (ESI) source of an APEX II instrument. The notably higher efficiency of ionization for compositional mapping of complex mixtures and feasibility for fragmentation analysis of components by sustained off-resonance irradiation collision-induced tandem mass spectrometry (SORI-CID MS2) has been demonstrated on a glycoconjugate mixture containing O-glycosylated sialylated peptides from urine of a patient suffering from a hereditary N-acetylhexosaminidase deficiency (Schindler's disease), previously analyzed by capillary-based nanoESI-FTICRMS, and of a healthy control person. Due to its potential to generate highly charged ionic species, reduce the in-source fragmentation, increase sensitivity, reproducibility and ionization efficiency, along with the ability to generate a sustained and constant electrospray, this method can be considered as a new platform for advanced glycomics.
Automation↗
Microbe manufacturers of semiconductors.
Synthesis of cadmium sulfide (CdS) semiconductor nanoparticles within a prokaryotic organism is reported for the first time by Sweeney et al. This paper demonstrates the utility of microorganisms to perform chemistries outside the scope of their "normal" metabolism and offers an environmentally benign synthesis of CdS nanoparticles.
Cadmium Compounds↗
Fully-automated chip-based nanoelectrospray tandem mass spectrometry of gangliosides from human cerebellum.
The introduction of chip-based electrospray (ESI) ion sources into biological mass spectrometry (MS) addressed the fundamental issue of how to analyze minute amounts of complex biological systems. The automation of sample delivery into the MS combined with the chip-based ESI allows for high quality bioanalysis in a high-throughput fashion. These advantages have already been demonstrated in proteomics, direct screening of drugs and drug discovery. As part of our continuing effort to implement automated chip-based mass spectrometry into the field of complex carbohydrate analysis, we hereby report the development of a chipESI MS and MS/MS methodology for the screening of gangliosides. A strategy to characterize a complex ganglioside mixture from human cerebellar tissue, by automated ESIchip-quadrupole time-of-flight (QTOF) MS and MS/MS is presented here. The feasibility of this method, and the general experimental requirements for automated chipESI MS analysis of these carbohydrate species is described.
Adult↗
Validating California teachers study self-reports of recent hospitalization: comparison with California hospital discharge data.
Determining an accurate method of obtaining complete morbidity data is a long-standing challenge for epidemiologists. The authors compared the accuracy and completeness of existing California hospital discharge data with self-reports of recent hospitalizations and surgeries from participants in the California Teachers Study. Self-reports were collected by questionnaire in 1997 from 91433 female teachers and administrators residing in California. Of the 13430 hospital discharge diagnoses identified for these women, cohort members reported 58%. Self-reporting was highest for neoplasms and musculoskeletal and connective tissue diseases and was most accurate for scheduled admissions, more recent admissions, longer lengths of stay, and less severe disorders. Hospitalizations for mental health and infectious disease were not as well reported. Among the 26383 self-reports-including outpatient surgeries, which are not captured by the hospital discharge database-confirmation was lower, as expected, especially for disorders of the nervous system and sense organs and skin and subcutaneous tissue. Confirmation was highest for childbirth admissions. The hospital discharge database was more specific, but the self-reports were more comprehensive, since many conditions are now treated in outpatient settings. The combination of self-reports and secondary medical records provides more accurate and complete morbidity data than does use of either source alone.
Aged↗
Constrained synthesis of cobalt oxide nanomaterials in the 12-subunit protein cage from Listeria innocua.
The protein cage of the 12-subunit ferritin-like protein from Listeria innocua has been utilized as a size and shape constrained reaction environment for the synthesis of two cobalt oxide minerals, Co(3)O(4) and Co(O)OH. Reaction of Co(II) with H(2)O(2) at pH 8.5 under either elevated temperature (65 degrees C) or ambient temperature (23 degrees C) resulted in the formation of cobalt oxide nanoparticles encapsulated within the protein cage. At elevated temperatures, Co(3)O(4) was formed while at lower temperature the oxyhydroxide Co(O)OH was found. Mineral particles, commensurate in size with the internal dimensions of the protein (5 nm), were imaged by transmission electron microscopy and shown to be surrounded by the intact protein cage. The minerals were investigated by electron diffraction and revealed a crystalline Co(3)O(4) phase and an amorphous Co(O)OH phase. Further investigation of these composite materials using size exclusion chromatography, gel electrophoresis, dynamic light scattering, and zeta potential measurements indicated that the mineral was encapsulated within the protein cage giving rise to properties of both the mineral and protein components.
Bacterial Proteins↗
Metal binding to cowpea chlorotic mottle virus using terbium(III) fluorescence.
Metals are thought to play a role in the structure of many viruses. The crystal structure of the T=3 icosahedral cowpea chlorotic mottle virus (CCMV) suggests the presence of 180 unique metal-binding sites in the assembled protein cage. Each of these sites is thought to involve the coordination of the metal by five amino acids contributed from two adjacent coat protein subunits. We have used fluorescence resonance energy transfer (FRET), from tryptophan residues proximal to the putative metal-binding sites, to probe Tb(III) binding to the virus. Binding of Tb(III) was investigated on the wild-type virus and a mutant where the RNA binding ability of the virus was removed. Tb(III) binding was observed both in the wild-type virus ( K(d)=19 microM) and the mutant ( K(d)=17 microM), as monitored by the increase in Tb(III) fluorescence (545 nm) and concomitant decrease in tryptophan fluorescence (342 nm). Competitive binding experiments showed Ca(II) to have about 100-fold less affinity for the binding sites ( K(d)=1.97 mM). This is the first direct evidence of metal binding to the putative metal-binding sites, originally suggested from the crystal structure of CCMV.
Binding Sites↗