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PubMed2005

Electron capture dissociation mass spectrometry in characterization of post-translational modifications.

Electron capture dissociation (ECD) represents a significant advance in tandem mass spectrometry for the identification and characterization of post-translational modifications (PTMs) of polypeptides. In comparison with the conventional fragmentation techniques, such as collisionally induced dissociation and infrared multi-photon dissociation, ECD provides more extensive sequence fragments, while allowing the labile modifications to remain intact during backbone fragmentation. This unique attribute offers ECD as an attractive alternative for detection and localization of PTMs. The success and rapid adoption of ECD recently led to the culmination of The 1st International Uppsala Symposium on Electron Capture Dissociation of Biomolecules and Related Phenomena (October 19-22, 2003, Stockholm, Sweden). Herein, we present a general overview of the ECD technique as well as selected applications in characterization of post-translationally modified polypeptides.

Electrons↗

PubMed2005

A simple immuno-capture ELISA to estimate rabies viral glycoprotein antigen in vaccine manufacture.

Rabies is an endemic, fatal zoonotic disease in the developing countries. Prevention and post-exposure therapy require safe and efficacious vaccines. The vaccine potency depends on the amount of immunogenic rabies viral glycoprotein antigen in the vaccine preparation. In order to estimate the rabies viral glycoprotein antigen, a specific monoclonal antibody was developed and used in an immuno-capture ELISA (IC-ELISA). The monoclonal antibody binds a conformational epitope on the natively folded rabies viral glycoprotein as indicated by specific, membrane fluorescence on unfixed, rabies virus infected murine neuroblastoma (MNA) cells and glycoprotein gene encoding plasmid transfected COS cells. In addition, the monoclonal antibody competes with and blocks a glycoprotein antigen site III binding monoclonal antibody (mAb-D1, Institut Pasteur, Paris, France). The monoclonal antibody was used in an IC-ELISA using an in-house standard to quantify the rabies viral glycoprotein antigen in 12 vaccine preparations with potency values ranging from 4 to 18 IU. The results indicated a good correlation with the NIH mouse potency assay (r=0.83). The immuno-capture ELISA described in this study can be used to quantify the immunogenic rabies viral glycoprotein antigen in the inactivated rabies viral antigen preparation in a simple and rapid format, which enables better vaccine formulation.

Animals↗

PubMed2005

A combinatorial approach to the selective capture of circulating malignant epithelial cells by peptide ligands.

Early detection is critical in the administration of definitive and curative therapy of cancer. However, current detection methods are ineffective at identifying the presence of circulating metastatic cancer cells in the blood because they typically sample only a relatively small volume of blood. One strategy for sampling larger blood volumes would be to capture circulating cells in vivo over an extended period of time. The development of such a method would be substantially facilitated by the identification of peptide ligands that bind selectively to metastatic cancer cells in the blood with high affinity. To identify such ligands a combinatorial peptide library was synthesized on polyethylene acrylamide (PEGA) resin and screened for binding to malignant epithelial cells. Using Biacore, cell binding assays were performed to demonstrate that peptides selected from PEGA bead screen can bind selectively to malignant epithelial cancer cells and not to circulating leukocytes under physiologic shear stress conditions. One peptide, with the sequence QMARIPKRLARH, was used to demonstrate selective labeling of malignant epithelial cells spiked in whole blood. When immobilized on appropriate surfaces, these peptides could be used in both in vivo and ex vivo cell separation devices to efficiently and selectively capture metastatic epithelial cancer cells from flowing blood.

Acrylamides↗

PubMed2006

The effect of electrochemical functionalization of Ti-alloy surfaces by aptamer-based capture molecules on cell adhesion.

To improve cell seeding efficiency and cytocompatibility, we designed a new coating material for scaffolds. We used aptamers, highly specific cell binding nucleic acids generated by combinatorial chemistry with an in vitro selection called systematic evolution of exponential enrichment (SELEX). In this study, we functionalized Ti-alloy surfaces to enhance cell adhesion. By coating the material with a cell specific aptamer, working as a capture molecule, we could improve the attachment of cells effectively and avoid the limitations of the currently available materials. Aptamers, immobilized by partial electrochemical entrapment in oxide layers on Ti-alloy surfaces were able to capture cells out of a flowing suspension rapidly. This model proves that surface immobilized aptamers can greatly enhance the attachment of seeded cells. This technology opens new perspectives towards clinical application of stem cell and tissue engineering strategies.

Animals↗

PubMed2006

Gene expression analysis of distinct populations of cells isolated from mouse and human inner ear FFPE tissue using laser capture microdissection--a technical report based on preliminary findings.

Laser Capture Microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. We first tested this method with sections of adult mouse inner ears and subsequently applied it to human inner ear sections. The morphology of the various cell types within the inner ear is well preserved in formalin fixed paraffin embedded (FFPE) sections, making it easier to identify cell types and their boundaries. Recovery of good quality RNA from FFPE sections can be challenging, however, recent studies in cancer research demonstrated that it is possible to carry out gene expression analysis of FFPE material. Thus, a method developed using mouse FFPE tissue can be applied to human archival temporal bones. This is important because the majority of human temporal bone banks have specimens preserved in formalin and a technique for retrospective analysis of human archival ear tissue is needed. We used mouse FFPE inner ear sections to procure distinct populations of cells from the various functional domains (organ of Corti, spiral ganglion, etc.) by LCM. RNA was extracted from captured cells, amplified, and assessed for quality. Expression of selected genes was tested by RT-PCR. In addition to housekeeping genes, we were able to detect cell type specific markers, such as Myosin 7a, p27(kip1) and neurofilament gene transcripts that confirmed the likely composition of cells in the sample. We also tested the method described above on FFPE sections from human crista ampullaris. These sections were approximately a year old. Populations of cells from the epithelium and stroma were collected and analyzed independently for gene expression. The method described here has potential use in many areas of hearing research. For example, following exposure to noise, ototoxic drugs or age, it would be highly desirable to analyze gene expression profiles of selected populations of cells within the organ of Corti or spiral ganglion cells rather than a mixed population of cells from whole inner ear tissue. Also, this method can be applied for analysis of human archival ear tissue.

Animals↗

PubMed2004

Covalent capture: a natural complement to self-assembly.

The utility of peptide self-assembly can be extended by covalent capture of these supramolecular materials. Disulfide bond formation, native chemical ligation, olefin metathesis, radical capture and oxidative lysine cross-linking have been used recently to help stabilize and characterize a variety of self-assembled peptides. These include natural peptides, proteins and protein mimics such as alpha-helical coiled coils, amyloid-like beta-sheet fibres, portions of p53, glutathione S-transferase and elastin as well as unnatural peptide constructs such as cyclic peptide nanotubes and cylindrical micelles of peptide amphiphiles.

Deamination↗

PubMed2005

Phosphoproteomic analysis with a solid-phase capture-release-tag approach.

A comprehensive study of global phosphorylation events in biological systems is critical. We report a chemistry-based capture-release-tag method for isolation of complex phospho-Ser/Thr-containing peptides by liquid beta-elimination combined with solid-phase Michael addition. The free thiol groups of 6-(mercapto-acetylamino)-hexanoic acid functionalized resin are used as immobilized Michael donors to capture dehydro-serine/threonine peptides. After an acid-mediated release step, phospho-peptides are labeled with a 6-(2-mercapto-acetylamine)-hexanoic amide tag at phosphorylated sites. We applied this method to analyze the phosphorylation status of microtubule-associated proteins. We find that a CDK5 substrate microtubule-associated protein 2 (MAP2) is phosphorylated on residues that are within a homologous region of Tau. The chemical method corroborates previous results and suggests that Tau and MAP2 may contain a CDK5 phosphorylation motif.

Amino Acid Sequence↗

PubMed2005

Structural effect of the in situ generated titania on its ability to oxidize and capture the gas-phase elemental mercury.

Structural effect of the in situ generated TiO(2) sorbent particle was examined for its ability to capture elemental mercury under UV irradiation in a simulated combustion flue gas. Titania particles were prepared by thermal gas-phase oxidation of Titanium (IV) isopropoxide (TTIP) using a high temperature electric furnace reactor. The structural characteristics of the in situ generated TiO(2) at various synthesis temperatures were investigated; size distribution and the geometric mean diameter were measured using a scanning mobility particle sizer, while fractal dimension and radius of gyration were evaluated from the transmission electron microscopy images. Results from the Hg(0) capture experiment show that with increasing titania synthesis temperature, the overall aggregate size increases and the morphology becomes more open-structured to gas-phase Hg(0) and UV light, resulting in the improved mercury removal capability.

Adsorption↗

PubMed2005

Quadrupole mass spectrometer operating in the electron-capture negative ion mode as detector for comprehensive two-dimensional gas chromatography.

A recently introduced rapid-scanning quadrupole mass spectrometer (qMS) with an electron-capture negative ion (ECNI) option, the Perkin-Elmer Clarus 500, was tested as a detector for comprehensive two-dimensional gas chromatography (GC x GC). The parameters influencing the data acquisition rate in the scan mode, such as scan time and inter-scan delay, and in the selected ion monitoring mode, such as dwell time and inter-channel delay, were evaluated. In the scan mode, good-quality mass spectra covering a range of 300 Da can be obtained at an acquisition rate of 23 Hz; in selected ion monitoring, an acquisition rate of 90Hz can be achieved when monitoring a single ion. Compared with electron ionisation, the use of electron-capture negative ionisation causes no extra peak broadening. As applications, mixtures of polychlorinated n-alkanes (PCAs), polybrominated diphenyl ethers (PBDEs) and polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) were analysed. The separation of PCAs based on their number of chlorine substituents was confirmed for the first time by using GC x GC-ECNI qMS in the scan mode and a significantly improved limit of detection was achieved for BDEs (10-150 fg injected) and CDD/Fs (10-700 fg injected) in the selected ion monitoring mode.

Gas Chromatography-Mass Spectrometry↗

PubMed2005

Solid-phase capture and release of arginine peptides by selective tagging and boronate affinity chromatography.

A method for the selection of arginine-containing peptides from a mixture by a solid phase capture and release technique is presented. The method is based on the covalent modification of the guanidine group of arginine with 2,3-butanedione and phenylboronic acid under alkaline conditions. Using polymeric materials with immobilised phenylboronic acid the arginine-peptides can be captured on a solid support while arginine-free peptides are not covalently bound and can be washed away. Finally, the arginine-peptides can be cleaved again from the boronic acid beads due to the reversibility of the reaction. The recovered peptides are then analysed by liquid chromatography-tandem mass spectrometry. The method was optimised with model peptides with regard to the non-specific binding of arginine-free peptides and quantitative cleavage of the label after the selection step. Using an adequate protocol, the applicability towards more complex samples was successfully tested with a tryptic digest of a mixture of three standard proteins.

Arginine↗

PubMed2004

Bodies capture attention when nothing is expected.

Functional neuroimaging research has shown that certain classes of visual stimulus selectively activate focal regions of visual cortex. Specifically, cortical areas that generally and selectively respond to faces (Kanwisher, N., McDermott, J., & Chun, M. M. (1997). The fusiform face area: a module in human extrastriate cortex specialized for face perception. Journal of Neuroscience, 17(11), 4302-4311; Puce, A., Allison, T., Asgari, M., Gore, J. C., & McCarthy, G. (1996). Differential sensitivity of human visual cortex to faces, letterstrings, and textures: a functional magnetic resonance imaging study. Journal of Neuroscience, 16(16), 5205-5215.) and to the human body (Downing, P. E., Jiang, Y., Shuman, M., & Kanwisher, N. (2001). A cortical area selective for visual processing of the human body. Science, 293(5539), 2470-2473.) have recently been described using fMRI. A parallel body of research has focused on the ability of faces to "capture" the focus of attention, compared to other kinds of objects (Lavie, N., Ro, T., & Russell, C. (2003). The role of perceptual load in processing distractor faces. Psychological Science, 14(5), 510-515; Ro, T., Russell, C., & Lavie, N. (2001). Changing faces: a detection advantage in the flicker paradigm. Psychological Science, 12(1), 94-99; Vuilleumier, P. (2000). Faces call for attention: evidence from patients with visual extinction. Neuropsychologia, 38(5), 693-700.). The present study uses Mack and Rock's "inattentional blindness" paradigm to investigate whether unexpected, task-irrelevant human body stimuli capture awareness when attention is occupied by a primary task (Mack, A., & Rock, I. (1998). Inattentional blindness. London: MIT Press). Silhouettes and stick figures of human bodies, and silhouettes of hands, were compared to control stimuli including object silhouettes, object stick figures, and scrambled silhouettes of bodies, body parts, and objects. Participants were significantly better able to detect a human figure relative to the control stimuli. These results suggest that the human body, like the face, may be prioritized for attentional selection. More generally, they are consistent with the proposal that the visual system assigns attentional priority to types of stimuli that are also represented in strongly selective cortical regions.

Attention↗

PubMed2006

Capturing the uncultivated majority.

The metagenomic analysis of environmental microbial communities continues to be a rapidly developing area of study. DNA isolation, the first step in capturing the uncultivated majority, has seen many advances in recent years. Protocols have been developed to distinguish DNA from live versus dead cells and to separate extracellular from intracellular DNA. Looking to increase our understanding of the role that members of a microbial community play in ecological processes, several techniques have been developed that are enabling greater in-depth analysis of environmental metagenomes. These include the development of environmental gene tags and the serial analysis of 16S rRNA gene sequence tags. In addition, new screening methods have been designed to select for specific functional genes within metagenomic libraries. Finally, new cultivation methods continue to be developed to improve our ability to capture a greater diversity of microorganisms within the environment.

DNA↗

PubMed2005

[Maternal inheritance in a subsocial spider: web for collective prey capturing of the young].

Construction of a safe refuge is the first step toward the evolution of social organization in spiders. In the case of the subsocial spider Amaurobius Jerox (Araneae, Amaurobiidae), the young remain in the natal nest after consuming their mother's body (matriphagy). The benefit that the young could obtain from the maternal web was investigated in their collective prey capture. The results demonstrated the adaptive value of the maternal web. The prey capturing activity was more effective when the young were allowed to stay on the maternal web than when they used the web constructed by themselves. Maternal web may also provide a better transmission of information on prey state and clutch mates' activities, because more spiderlings were recruited to contact and hold the prey in a shorter period of time than when they were on a spiderling-constructed web. The young's inheritance of the maternal nest is probably one of the crucial ways in which the mother influences the post-maternal social period of the young.

Animals↗

PubMed2006

Compartmentalized PKA signaling events are required for synaptic tagging and capture during hippocampal late-phase long-term potentiation.

Synaptic plasticity, the activity-dependent change in the strength of neuronal connections, is a proposed cellular mechanism of memory storage that is critically regulated by protein kinases such as cAMP-dependent protein kinase (PKA). Despite the fact that a neuron contains thousands of synapses, the expression of synaptic plasticity can be specific to subsets of synapses. This is surprising because signal transduction pathways underlying synaptic plasticity involve diffusible second messenger molecules such as cAMP and diffusible proteins such as the catalytic subunit of PKA. One way in which this specificity can be achieved is by the localization of signal transduction molecules to specific subcellular domains. Spatial compartmentalization of PKA signaling is achieved via binding to A kinase-anchoring proteins (AKAPs). We report here that pharmacological inhibition of PKA anchoring impairs synaptically activated late-phase long-term potentiation (L-LTP) in hippocampal slices. In contrast, potentiation that is induced by the pharmacological activation of the cAMP/PKA pathway, which can potentially affect all synapses within the neuron, is not impaired by inhibition of PKA anchoring. These results suggest that PKA anchoring may be particularly important for events that occur at the synapse during the induction of L-LTP, such as synaptic tagging and capture. Indeed, our results demonstrate that blocking PKA anchoring impairs synaptic tagging and capture. Thus our data highlight the idea that PKA anchoring may allow for specific populations of synapses to change in synaptic strength in the face of plasticity-related transcription that is cell-wide.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Understanding capture detection.

Automatic capture detection systems are currently available in several cardiac pacing devices. All current systems use low-polarization electrodes and no beat to beat detection system is available for all types of electrodes. In addition the success ratio for currently available systems is not always 100%. Failure to detect capture reliably is often related to the behaviour of the electrode-tissue interface under different circumstances. Pacemaker electrodes can be considered electrochemical cells with complicated characteristics depending on time, temperature and electrical charge. This electrochemical cell is disturbed when a charge is transferred across the electrode-tissue interface during pacing. Several measures can be taken in order to minimise this disturbance or pace polarization artefact (PPA) including the use of high active surface area electrodes and application of tri-phasic pacing pulses. Another factor influencing detection of evoked potentials is the input circuit of the pacemaker affecting the PPA and the evoked response. Positive PPAs can be falsely interpreted as evoked potentials due to the undershoot of the second order filters applied in modern cardiac pacemakers. This paper explains the behaviour of the interface between the electrode and the cardiac tissue in combination with the pacemaker output circuits and input amplifiers under different circumstances.

Cardiac Pacing, Artificial↗

PubMed2004

Evaluation of a standardized F1 capsular antigen capture ELISA test kit for the rapid diagnosis of plague.

Rapid detection of soluble F1 capsular antigen in serum, bubo fluid or urine of patients proved to be a valuable tool in the presumptive diagnosis of plague. We evaluated a F1 capsular antigen capture ELISA resembling a commercially available test kit. The minimal detectable concentration was 4 ng/ml. The specificity was 100% when investigating 47 sera from healthy Malagasy subjects and 98.4% when 365 sera from German blood donors were studied. Sensitivity was determined on sera (n=11) and buboes (n=18) from bacteriologically confirmed Malagasy plague patients. Sensitivity was 90.1% for serum and 100% for buboes. A standardized F1 capsular antigen capture ELISA test kit might be well suited for the early detection of plague particularly in non-endemic areas where clinical microbiological laboratories have only limited access to alternative techniques for rapid identification of Yersinia pestis.

Antibodies, Monoclonal↗

PubMed2005

Hyperkalemia induced failure of atrial and ventricular pacemaker capture.

Hyperkalemia is a life threatening metabolic condition. The common risk factors for hyperkalemia include renal insufficiency, use of angiotensin converting enzyme inhibitors, potassium supplementation and excessive consumption of potassium containing diet. A mild to moderate increase in serum potassium causes an increase in myocardial excitability, but further increase leads to impaired myocardial responsiveness, including that to pacing stimulation. Hyperkalemia has been reported to cause failure of atrial capture due to pacemaker exit block. We report a case where hyperkalemia resulted in failure of both the atrial and the ventricular pacemaker capture.

Electrocardiography↗

PubMed2022

Direct capture and sequencing reveal ultra-short single-stranded DNA in biofluids.

Cell-free DNA (cfDNA) has become the predominant analyte of liquid biopsy; however, recent studies suggest the presence of subnucleosomal-sized DNA fragments in circulation that are likely single-stranded. Here, we report a method called direct capture and sequencing (DCS) tailored to recover such fragments from biofluids by directly capturing them using short degenerate probes followed by single strand-based library preparation and next-generation sequencing. DCS revealed a new DNA population in biofluids, named ultrashort single-stranded DNA (ussDNA). Evaluation of the size distribution and abundance of ussDNA manifested generality of its presence in humans, animal species, and plants. In humans, red blood cells were found to contain abundant ussDNA; plasma-derived ussDNA exhibited modal size at 50 nt. This work reports the presence of an understudied DNA population in circulation, and yet more work is awaiting to study its generation mechanism, tissue of origin, disease implications, etc.

Biological sciences↗