Quantitative proteome analysis in cardiovascular physiology and pathology. I. Data processing

Methodological evaluation of the proteomic analysis of cardiovascular-tissue material has been performed with a special emphasis on establishing examinations that allow reliable quantitative analysis of silver-stained readouts. Reliability, reproducibility, robustness and linearity were addressed and clarified. In addition, several types of normalization procedures were evaluated and new approaches are proposed. It has been found that the silver-stained readout offers a convenient approach for quantitation if a linear range for gel loading is defined. In addition, a broad range of a 10-fold input (loading 20-200 microg per gel) fulfills the linearity criteria, although at the lowest input (20 microg) a portion of protein species will remain undetected. The method is reliable and reproducible within a range of 65-200 microg input. The normalization procedure using the sum of all spot intensities from a silver-stained 2D pattern has been shown to be less reliable than other approaches, namely, normalization through median or through involvement of interquartile range. A special refinement of the normalization through virtual segmentation of pattern, and calculation of normalization factor for each stratum provides highly satisfactory results. The presented results not only provide evidence for the usefulness of silver-stained gels for quantitative evaluation, but they are directly applicable to the research endeavor of monitoring alterations in cardiovascular pathophysiology.

Hierarchical Markov Random Fields Applied to Model Soft Tissue Deformations on Graphics Hardware

Many methodologies dealing with prediction or simulation of soft tissue deformations on medical image data require preprocessing of the data in order to produce a different shape representation that complies with standard methodologies, such as mass–spring networks, finite element method s (FEM). On the other hand, methodologies working directly on the image space normally do not take into account mechanical behavior of tissues and tend to lack physics foundations driving soft tissue deformations. This chapter presents a method to simulate soft tissue deformations based on coupled concepts from image analysis and mechanics theory. The proposed methodology is based on a robust stochastic approach that takes into account material properties retrieved directly from the image, concepts from continuum mechanics and FEM. The optimization framework is solved within a hierarchical Markov random field (HMRF) which is implemented on the graphics processor unit (GPU See Graphics processing unit ).

Virtual tissue alignment and cutting plane definition--a new method to obtain optimal longitudinal histological sections.

Histomorphometric evaluation of the buccal aspects of periodontal tissues in rodents requires reproducible alignment of maxillae and highly precise sections containing central sections of buccal roots; this is a cumbersome and technically sensitive process due to the small specimen size. The aim of the present report is to describe and analyze a method to transfer virtual sections of micro-computer tomographic (CT)-generated image stacks to the microtome for undecalcified histological processing and to describe the anatomy of the periodontium in rat molars. A total of 84 undecalcified sections of all buccal roots of seven untreated rats was analyzed. The accuracy of section coordinate transfer from virtual micro-CT slice to the histological slice, right-left side differences and the measurement error for linear and angular measurements on micro-CT and on histological micrographs were calculated using the Bland-Altman method, interclass correlation coefficient and the method of moments estimator. Also, manual alignment of the micro-CT-scanned rat maxilla was compared with multiplanar computer-reconstructed alignment. The supra alveolar rat anatomy is rather similar to human anatomy, whereas the alveolar bone is of compact type and the keratinized gingival epithelium bends apical to join the junctional epithelium. The high methodological standardization presented herein ensures retrieval of histological slices with excellent display of anatomical microstructures, in a reproducible manner, minimizes random errors, and thereby may contribute to the reduction of number of animals needed.

Ceramic foam plates: a new tool for processing fresh radical prostatectomy specimens

Procurement of fresh tissue of prostate cancer is critical for biobanking and generation of xenograft models as an important preclinical step towards new therapeutic strategies in advanced prostate cancer. However, handling of fresh radical prostatectomy specimens has been notoriously challenging given the distinctive physical properties of prostate tissue and the difficulty to identify cancer foci on gross examination. Here, we have developed a novel approach using ceramic foam plates for processing freshly cut whole mount sections from radical prostatectomy specimens without compromising further diagnostic assessment. Forty-nine radical prostatectomy specimens were processed and sectioned from the apex to the base in whole mount slices. Putative carcinoma foci were morphologically verified by frozen section analysis. The fresh whole mount slices were then laid between two ceramic foam plates and fixed overnight. To test tissue preservation after this procedure, formalin-fixed and paraffin-embedded whole mount sections were stained with hematoxylin and eosin (H&E) and analyzed by immunohistochemistry, fluorescence, and silver in situ hybridization (FISH and SISH, respectively). There were no morphological artifacts on H&E stained whole mount sections from slices that had been fixed between two plates of ceramic foam, and the histological architecture was fully retained. The quality of immunohistochemistry, FISH, and SISH was excellent. Fixing whole mount tissue slices between ceramic foam plates after frozen section examination is an excellent method for processing fresh radical prostatectomy specimens, allowing for a precise identification and collection of fresh tumor tissue without compromising further diagnostic analysis.

Thermal processing of bone: in vitro response of mesenchymal cells to bone-conditioned medium

The autoclaving, pasteurization, and freezing of bone grafts to remove bacteria and viruses, and for preservation, respectively, is considered to alter biological properties during graft consolidation. Fresh bone grafts release paracrine-like signals that are considered to support tissue regeneration. However, the impact of the autoclaving, pasteurization, and freezing of bone grafts on paracrine signals remains unknown. Therefore, conditioned medium was prepared from porcine cortical bone chips that had undergone thermal processing. The biological properties of the bone-conditioned medium were assessed by examining the changes in expression of target genes in oral fibroblasts. The data showed that conditioned medium obtained from bone chips that had undergone pasteurization and freezing changed the expression of adrenomedullin, pentraxin 3, BTB/POZ domain-containing protein 11, interleukin 11, NADPH oxidase 4, and proteoglycan 4 by at least five-fold in oral fibroblasts. Bone-conditioned medium obtained from autoclaved bone chips, however, failed to change the expression of the respective genes. Also, when bone-conditioned medium was prepared from fresh bone chips, autoclaving blocked the capacity of bone-conditioned medium to modulate gene expression. These in vitro results suggest that pasteurization and freezing of bone grafts preserve the release of biologically active paracrine signals, but autoclaving does not. Copyright © 2015 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. KEYWORDS: allogeneic bone; augmentation; autoclaving; autologous bone; bone bank; bone grafts; bone regeneration; bone supernatant; bone-conditioned medium; freezing; pasteurization

Variable effects of the conserved RNA hairpin element upon 3' end processing of histone pre-mRNA in vitro.

We have studied the requirements for efficient histone-specific RNA 3' processing in nuclear extract from mammalian tissue culture cells. Processing is strongly impaired by mutations in the pre-mRNA spacer element that reduce the base-pairing potential with U7 RNA. Moreover, by exchanging the hairpin and spacer elements of two differently processed H4 genes, we find that this difference is exclusively due to the spacer element. Finally, processing is inhibited by the addition of competitor RNAs, if these contain a wild-type spacer sequence, but not if their spacer element is mutated. Conversely, the importance of the hairpin for histone RNA 3' processing is highly variable: A hairpin mutant of the H4-12 gene is processed with almost wild-type efficiency in extract from K21 mouse mastocytoma cells but is strongly affected in HeLa cell extract, whereas an identical hairpin mutant of the H4-1 gene is affected in both extracts. The hairpin defect of H4-12-specific RNA in HeLa cells can be overcome by a compensatory mutation that increases the base complementarity to U7 snRNA. Very similar results were also obtained in RNA competition experiments: processing of H4-12-specific RNA can be competed by RNA carrying a wild-type hairpin element in extract from HeLa, but not K21 cells, whereas processing of H4-1-specific RNA can be competed in both extracts. With two additional histone genes we obtained results that were in one case intermediate and in the other similar to those obtained with H4-1. These results suggest that hairpin binding factor(s) can cooperatively support the ability of U7 snRNPs to form an active processing complex, but is(are) not directly involved in the processing mechanism.

Atlas-free Brain Tissue Segmentation Using a Single T1-weighted MRI Acquisition

Many studies investigating the aging brain or disease-induced brain alterations rely on accurate and reproducible brain tissue segmentation. Being a preliminary processing step prior to the segmentation, reliableskull-stripping the removal ofnon-brain tissue is also crucial for all later image assessment. Typically, segmentation algorithms rely on an atlas i.e. pre-segmented template data. Brain morphology, however, differs considerably depending on age, sex and race. In addition, diseased brains may deviate significantly from the atlas information typically gained from healthy volunteers. The imposed prior atlas information can thus lead to degradation of segmentation results. The recently introduced MP2RAGE sequence provides a bias-free T1 contrast with heavily reduced T2*- and PD-weighting compared to the standard MP-RAGE [1]. To this end, it acquires two image volumes at different inversion times in one acquisition, combining them to a uniform, i.e. homogenous image. In this work, we exploit the advantageous contrast properties of the MP2RAGE and combine it with a Dixon (i.e. fat-water separation) approach. The information gained by the additional fat image of the head considerably improves the skull-stripping outcome [2]. In conjunction with the pure T1 contrast of the MP2RAGE uniform image, we achieve robust skull-stripping and brain tissue segmentation without the use of an atlas

Image processing methods for human brain connectivity analysis from in-vivo diffusion MRI

The structural connectivity of the brain is considered to encode species-wise and subject-wise patterns that will unlock large areas of understanding of the human brain. Currently, diffusion MRI of the living brain enables to map the microstructure of tissue, allowing to track the pathways of fiber bundles connecting the cortical regions across the brain. These bundles are summarized in a network representation called connectome that is analyzed using graph theory. The extraction of the connectome from diffusion MRI requires a large processing flow including image enhancement, reconstruction, segmentation, registration, diffusion tracking, etc. Although a concerted effort has been devoted to the definition of standard pipelines for the connectome extraction, it is still crucial to define quality assessment protocols of these workflows. The definition of quality control protocols is hindered by the complexity of the pipelines under test and the absolute lack of gold-standards for diffusion MRI data. Here we characterize the impact on structural connectivity workflows of the geometrical deformation typically shown by diffusion MRI data due to the inhomogeneity of magnetic susceptibility across the imaged object. We propose an evaluation framework to compare the existing methodologies to correct for these artifacts including whole-brain realistic phantoms. Additionally, we design and implement an image segmentation and registration method to avoid performing the correction task and to enable processing in the native space of diffusion data. We release PySDCev, an evaluation framework for the quality control of connectivity pipelines, specialized in the study of susceptibility-derived distortions. In this context, we propose Diffantom, a whole-brain phantom that provides a solution to the lack of gold-standard data. The three correction methodologies under comparison performed reasonably, and it is difficult to determine which method is more advisable. We demonstrate that susceptibility-derived correction is necessary to increase the sensitivity of connectivity pipelines, at the cost of specificity. Finally, with the registration and segmentation tool called regseg we demonstrate how the problem of susceptibility-derived distortion can be overcome allowing data to be used in their original coordinates. This is crucial to increase the sensitivity of the whole pipeline without any loss in specificity.

Tsetse thrombin inhibitor: Bloodmeal-induced expression of an anticoagulant in salivary glands and gut tissue of Glossina morsitans morsitans

The tsetse thrombin inhibitor, a potent and specific low molecular mass (3,530 Da) anticoagulant peptide, was purified previously from salivary gland extracts of Glossina morsitans morsitans (Diptera: Glossinidae). A 303-bp coding sequence corresponding to the inhibitor has now been isolated from a tsetse salivary gland cDNA library by using degenerate oligonucleotide probes. The full-length cDNA contains a 26-bp untranslated segment at its 5′ end, followed by a 63-bp sequence corresponding to a putative secretory signal peptide. A 96-bp segment codes for the mature tsetse thrombin inhibitor, whose predicted molecular weight matches that of the purified native protein. Based on its lack of homology to any previously described family of molecules, the tsetse thrombin inhibitor appears to represent a unique class of naturally occurring protease inhibitors. Recombinant tsetse thrombin inhibitor expressed in Escherichia coli and the chemically synthesized peptide are both substantially less active than the purified native protein, suggesting that posttranslational modification(s) may be necessary for optimal inhibitory activity. The tsetse thrombin inhibitor gene, which is present as a single copy in the tsetse genome, is expressed at high levels in salivary glands and midguts of adult tsetse flies, suggesting a possible role for the anticoagulant in both feeding and processing of the bloodmeal.

Activation of target-tissue immune-recognition molecules by double-stranded polynucleotides

Abnormal expression of major histocompatibility complex (MHC) class I and class II in various tissues is associated with autoimmune disease. Autoimmune responses can be triggered by viral infections or tissue injuries. We show that the ability of a virus or a tissue injury to increase MHC gene expression is duplicated by any fragment of double-stranded (ds) DNA or dsRNA introduced into the cytoplasm of nonimmune cells. Activation is sequence-independent, is induced by ds polynucleotides as small as 25 bp in length, and is not duplicated by single-stranded polynucleotides. In addition to causing abnormal MHC expression, the ds nucleic acids increase the expression of genes necessary for antigen processing and presentation: proteasome proteins (e.g., LMP2), transporters of antigen peptides; invariant chain, HLA-DM, and the costimulatory molecule B7.1. The mechanism is different from and additive to that of γ-interferon (γIFN), i.e., ds polynucleotides increase class I much more than class II, whereas γIFN increases class II more than class I. The ds nucleic acids also induce or activate Stat1, Stat3, mitogen-activated protein kinase, NF-κB, the class II transactivator, RFX5, and the IFN regulatory factor 1 differently from γIFN. CpG residues are not responsible for this effect, and the action of the ds polynucleotides could be shown in a variety of cell types in addition to thyrocytes. We suggest that this phenomenon is a plausible mechanism that might explain how viral infection of tissues or tissue injury triggers autoimmune disease; it is potentially relevant to host immune responses induced during gene therapy.

Caspase-1 is activated in neural cells and tissue with amyotrophic lateral sclerosis-associated mutations in copper-zinc superoxide dismutase

The mechanism by which mutations in the superoxide dismutase (SOD1) gene cause motor neuron degeneration in familial amyotrophic lateral sclerosis (ALS) is unknown. Recent reports that neuronal death in SOD1-familial ALS is apoptotic have not documented activation of cell death genes. We present evidence that the enzyme caspase-1 is activated in neurons expressing mutant SOD1 protein. Proteolytic processing characteristic of caspase-1 activation is seen both in spinal cords of transgenic ALS mice and neurally differentiated neuroblastoma (line N2a) cells with SOD1 mutations. This activation of caspase-1 is enhanced by oxidative challenge (xanthine/xanthine oxidase), which triggers cleavage and secretion of the interleukin 1β converting enzyme substrate, pro-interleukin 1β, and induces apoptosis. This N2a culture system should be an instructive in vitro model for further investigation of the proapoptotic properties of mutant SOD1.

Nucellain, a Barley Homolog of the Dicot Vacuolar-Processing Protease, Is Localized in Nucellar Cell Walls1

The nucellus is a complex maternal grain tissue that embeds and feeds the developing cereal endosperm and embryo. Differential screening of a barley (Hordeum vulgare) cDNA library from 5-d-old ovaries resulted in the isolation of two cDNA clones encoding nucellus-specific homologs of the vacuolar-processing enzyme of castor bean (Ricinus communis). Based on the sequence of these barley clones, which are called nucellains, a homolog from developing corn (Zea mays) grains was also identified. In dicots the vacuolar-processing enzyme is believed to be involved in the processing of vacuolar storage proteins. RNA-blot and in situ-hybridization analyses detected nucellain transcripts in autolysing nucellus parenchyma cells, in the nucellar projection, and in the nucellar epidermis. No nucellain transcripts were detected in the highly vacuolate endosperm or in the other maternal tissues of developing grains such as the testa or the pericarp. Using an antibody raised against castor bean vacuolar-processing protease, a single polypeptide was recognized in protein extracts from barley grains. Immunogold-labeling experiments with this antibody localized the nucellain epitope not in the vacuoles, but in the cell walls of all nucellar cell types. We propose that nucellain plays a role in processing and/or turnover of cell wall proteins in developing cereal grains.

Fluorescent light-induced chromatid breaks distinguish Alzheimer disease cells from normal cells in tissue culture.

The neurodegeneration and amyloid deposition of sporadic Alzheimer disease (AD) also occur in familial AD and in all trisomy-21 Down syndrome (DS) patients, suggesting a common pathogenetic mechanism. We investigated whether defective processing of damaged DNA might be that mechanism, as postulated for the neurodegeneration in xeroderma pigmentosum, a disease with defective repair not only of UV radiation-induced, but also of some oxygen free radical-induced, DNA lesions. We irradiated AD and DS skin fibroblasts or blood lymphocytes with fluorescent light, which is known to cause free radical-induced DNA damage. The cells were then treated with either beta-cytosine arabinoside (araC) or caffeine, and chromatid breaks were quantified. At least 28 of 31 normal donors and 10 of 11 donors with nonamyloid neurodegenerations gave normal test results. All 12 DS, 11 sporadic AD, and 16 familial AD patients tested had abnormal araC and caffeine tests, as did XP-A cells. In one of our four AD families, an abnormal caffeine test was found in all 10 afflicted individuals (including 3 asymptomatic when their skin biopsies were obtained) and in 8 of 11 offspring at a 50% risk for AD. Our tests could prove useful in predicting inheritance of familial AD and in supporting, or rendering unlikely, the diagnosis of sporadic AD in patients suspected of having the disease.

Two alternative processing pathways for a preprohormone: a bioactive form of secretin.

An N-terminally 9-residue elongated form of secretin, secretin-(-9 to 27) amide, was isolated from porcine intestinal tissue and characterized. Current knowledge about peptide processing sites does not allow unambiguous prediction of the signal peptide cleavage site in preprosecretin but suggests cleavage in the region of residues -10 to -14 counted upstream from the N terminus of the hormone. However, the structure of the isolated peptide suggests that the cleavage between the signal peptide and the N-terminal propeptide occurs at the C-terminal side of residue -10. Moreover, the isolated peptide demonstrates that secretin can be fully processed C-terminally prior to the final N-terminal cleavage. The results from this report, and those from earlier studies, where C-terminally elongated variants were isolated, show that the processing of the secretin precursor may proceed by one of two alternative pathways, in which either of the two ends is processed first. The bioactivity of the N-terminally extended peptide on exocrine pancreatic secretion was lower than that of secretin, indicating the importance of the finally processed free N terminus of the hormone for interaction with secretin receptors.