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2.
Sci Eng Ethics ; 21(4): 1049-64, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25150847

ABSTRACT

Here we identify approximately 40,000 healthy human volunteers who were intentionally exposed to infectious pathogens in clinical research studies dating from late World War II to the early 2000s. Microbial challenge experiments continue today under contemporary human subject research requirements. In fact, we estimated 4,000 additional volunteers who were experimentally infected between 2010 and the present day. We examine the risks and benefits of these experiments and present areas for improvement in protections of participants with respect to safety. These are the absence of maximum limits to risk and the potential for institutional review boards to include questionable benefits to subjects and society when weighing the risks and benefits of research protocols. The lack of a duty of medical care by physician-investigators to research subjects is likewise of concern. The transparency of microbial challenge experiments and the safety concerns raised in this work may stimulate further dialogue on the risks to participants of human experimentation.


Subject(s)
Biomedical Research/ethics , Healthy Volunteers , Infections , Intention , Research Design , Research Subjects , Safety , Ethics Committees, Research , Ethics, Medical , Ethics, Research , Human Experimentation/ethics , Humans , Informed Consent , Research Personnel/ethics , Risk , Risk Assessment
3.
J Proteomics Bioinform ; 7(6): 151-157, 2014 Jun 24.
Article in English | MEDLINE | ID: mdl-25049470

ABSTRACT

Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE tissue. Our laboratory has taken a mechanistic approach to developing improved protein extraction protocols, by first studying the reactions of formaldehyde with proteins and ways to reverse these reactions, then applying this approach to a model system called a "tissue surrogate", which is a gel formed by treating high concentrations of cytoplasmic proteins with formaldehyde, and finally FFPE mouse liver tissue. Our studies indicate that elevated pressure improves the recovery of proteins from FFPE tissue surrogates by hydrating and promoting solubilization of highly aggregated proteins allowing for the subsequent reversal (by hydrolysis) of formaldehyde-induced protein adducts and cross-links. When FFPE mouse liver was extracted using heat and elevated pressure, there was a 4-fold increase in protein extraction efficiency and up to a 30-fold increase in the number of non-redundant proteins identified by mass spectrometry, compared to matched tissue extracted with heat alone. More importantly, the number of non-redundant proteins identified in the FFPE tissue was nearly identical to that of the corresponding frozen tissue.

4.
J Cancer ; 5(2): 115-24, 2014.
Article in English | MEDLINE | ID: mdl-24494029

ABSTRACT

Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) have been validated at the highest level of evidence as clinical biomarkers of prognosis in breast cancer. The American Society of Clinical Oncology recommends using uPA and PAI-1 levels in breast tumors for deciding whether patients with newly diagnosed node-negative breast cancer can forgo adjuvant chemotherapy. The sole validated method for quantifying uPA and PAI-1 levels in breast tumor tissue is a colorimetric ELISA assay that takes 3 days to complete and requires 100-300 mg of fresh or frozen tissue. In this study we describe a new assay method for quantifying PAI-1 levels in human breast tumor tissue. This assay combines pressure-cycling technology to extract PAI-1 from breast tumor tissue with a highly sensitive liposome polymerase chain reaction immunoassay for quantification of PAI-1 in the tissue extract. The new PAI-1 assay method reduced the total assay time to one day and improved assay sensitivity and dynamic range by >100, compared to ELISA.

5.
Expert Rev Proteomics ; 10(4): 389-400, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23992421

ABSTRACT

Archival formalin-fixed, paraffin-embedded (FFPE) tissue and their associated diagnostic records represent an invaluable source of retrospective proteomic information on diseases for which the clinical outcome and response to treatment are known. However, analysis of archival FFPE tissues by high-throughput proteomic methods has been hindered by the adverse effects of formaldehyde fixation and subsequent tissue histology. This review examines recent methodological advances for extracting proteins from FFPE tissue suitable for proteomic analysis. These methods, based largely upon heat-induced antigen retrieval techniques borrowed from immunohistochemistry, allow at least a qualitative analysis of the proteome of FFPE archival tissues. The authors also discuss recent advances in the proteomic analysis of FFPE tissue; including liquid-chromatography tandem mass spectrometry, reverse phase protein microarrays and imaging mass spectrometry.


Subject(s)
Proteomics/methods , Tissue Embedding/methods , Tissue Fixation/methods , Animals , Formaldehyde , Humans , Mass Spectrometry/methods , Paraffin
6.
Proteomics Clin Appl ; 7(3-4): 264-72, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23339100

ABSTRACT

Tissue-based proteomic approaches (tissue proteomics) are essential for discovering and evaluating biomarkers for personalized medicine. In any proteomics study, the most critical issue is sample extraction and preparation. This problem is especially difficult when recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, improving and standardizing protein extraction from FFPE tissue is a critical need because of the millions of archival FFPE tissues available in tissue banks worldwide. Recent progress in the application of heat-induced antigen retrieval principles for protein extraction from FFPE tissue has resulted in a number of published FFPE tissue proteomics studies. However, there is currently no consensus on the optimal protocol for protein extraction from FFPE tissue or accepted standards for quantitative evaluation of the extracts. Standardization is critical to ensure the accurate evaluation of FFPE protein extracts by proteomic methods such as reverse phase protein arrays, which is now in clinical use. In our view, complete solubilization of FFPE tissue samples is the best way to achieve the goal of standardizing the recovery of proteins from FFPE tissues. However, further studies are recommended to develop standardized protein extraction methods to ensure quantitative and qualitative reproducibility in the recovery of proteins from FFPE tissues.


Subject(s)
Fixatives/chemistry , Formaldehyde/chemistry , Paraffin Embedding/methods , Paraffin/chemistry , Proteome/analysis , Biomarkers/analysis , Humans , Reproducibility of Results
7.
J Nanobiotechnology ; 10(1): 26, 2012 Jun 22.
Article in English | MEDLINE | ID: mdl-22726242

ABSTRACT

BACKGROUND: The accurate quantification of antigens at low concentrations over a wide dynamic range is needed for identifying biomarkers associated with disease and detecting protein interactions in high-throughput microarrays used in proteomics. Here we report the development of an ultrasensitive quantitative assay format called immunoliposome polymerase chain reaction (ILPCR) that fulfills these requirements. This method uses a liposome, with reporter DNA encapsulated inside and biotin-labeled polyethylene glycol (PEG) phospholipid conjugates incorporated into the outer surface of the liposome, as a detection reagent. The antigenic target is immobilized in the well of a microplate by a capture antibody and the liposome detection reagent is then coupled to a biotin-labeled second antibody through a NeutrAvidin bridge. The liposome is ruptured to release the reporter DNA, which serves as a surrogate to quantify the protein target using real-time PCR. RESULTS: A liposome detection reagent was prepared, which consisted of a population of liposomes ~120 nm in diameter with each liposome possessing ~800 accessible biotin receptors and ~220 encapsulated reporters. This liposome detection reagent was used in an assay to quantify the concentration of carcinoembryonic antigen (CEA) in human serum. This ILPCR assay exhibited a linear dose-response curve from 10-10 M to 10-16 M CEA. Within this range the assay coefficient of variance was <6 % for repeatability and <2 % for reproducibility. The assay detection limit was 13 fg/mL, which is 1,500-times more sensitive than current clinical assays for CEA. An ILPCR assay to quantify HIV-1 p24 core protein in buffer was also developed. CONCLUSIONS: The ILPCR assay has several advantages over other immuno-PCR methods. The reporter DNA and biotin-labeled PEG phospholipids spontaneously incorporate into the liposomes as they form, simplifying preparation of the detection reagent. Encapsulation of the reporter inside the liposomes allows nonspecific DNA in the assay medium to be degraded with DNase I prior to quantification of the encapsulated reporter by PCR, which reduces false-positive results and improves quantitative accuracy. The ability to encapsulate multiple reporters per liposome also helps overcome the effect of polymerase inhibitors present in biological specimens. Finally, the biotin-labeled liposome detection reagent can be coupled through a NeutrAvidin bridge to a multitude of biotin-labeled probes, making ILPCR a highly generic assay system.


Subject(s)
Carcinoembryonic Antigen/blood , Liposomes/chemistry , Polymerase Chain Reaction/methods , Antibodies, Immobilized/immunology , Avidin/chemistry , Biotin/chemistry , Carcinoembryonic Antigen/genetics , HIV Core Protein p24/analysis , HIV-1/metabolism , Humans , Polyethylene Glycols/chemistry , Rhodamines/chemistry
8.
J Proteome Res ; 11(4): 2602-8, 2012 Apr 06.
Article in English | MEDLINE | ID: mdl-22352854

ABSTRACT

Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE mouse liver. When FFPE mouse liver was extracted using heat and elevated pressure, there was a 4-fold increase in protein extraction efficiency, a 3-fold increase in the extraction of intact proteins, and up to a 30-fold increase in the number of nonredundant proteins identified by mass spectrometry, compared to matched tissue extracted with heat alone. More importantly, the number of nonredundant proteins identified in the FFPE tissue was nearly identical to that of matched fresh-frozen tissue.


Subject(s)
Proteins/isolation & purification , Proteome/isolation & purification , Proteomics/methods , Animals , Formaldehyde , Hot Temperature , Hydrostatic Pressure , Liver/chemistry , Mice , Paraffin Embedding , Proteins/analysis , Proteins/chemistry , Proteome/analysis , Proteome/chemistry
9.
J Mol Diagn ; 13(6): 687-94, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21884819

ABSTRACT

The RNA isolated from FFPE tissues is of poor quality and quantity. Other studies have indicated that formaldehyde fixation or the duration of storage of tissue blocks accounted for RNA damage. Herein we report a third source of harm to RNA: embedding in warm paraffin. RNA bound to oligo(dT)-conjugated magnetic beads (an mRNA model) and total cellular RNA pellets were passed through formalin, graded ethanols, xylene, paraffin, and a formaldehyde demodification step. The mRNA model yielded at least 1550 bp amplicons at RT-PCR at each step of processing except paraffin, which yielded no more than 750 bp amplicons regardless of paraffin formulation or transition solvent. Quantitative RT-PCR on paraffinized RNA suggested a 1400-fold or more decrease in amplifiable RNA when compared with control. Compared with earlier processing steps, formalin-fixed paraffinized total cellular RNA produced only high-molecular-weight RNA and insoluble aggregates. These species were reproduced by heating RNA in hydrocarbon solvent at 60°C for 1 hour. Quantitative RT-PCR on paraffinized RNA suggested an at least 10- to 160-fold decrease in amplifiable RNA compared to controls. The data implicate paraffin embedding as primarily responsible for the high-molecular-weight RNA aggregates, reduced yields of RNA, and poor quality of RNA isolated from these chemical models of FFPE tissues.


Subject(s)
Paraffin Embedding , RNA Stability , RNA/chemistry , Tissue Fixation/methods , Denaturing Gradient Gel Electrophoresis , Formaldehyde , HeLa Cells , Humans , Reverse Transcriptase Polymerase Chain Reaction
10.
Magn Reson Imaging ; 29(9): 1244-54, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21920685

ABSTRACT

Magnetic resonance imaging (MRI) studies of tissue engineered constructs prior to implantation clearly demonstrate the utility of the MRI technique for studying the bone formation process. To test the utility of our MRI protocols for explant studies, we present a novel test platform in which osteoblast-seeded scaffolds were implanted on the chorioallantoic membrane of a chick embryo. Scaffolds from the following experimental groups were examined by high-resolution MRI: (a) cell-seeded implanted scaffolds (CIM), (b) unseeded implanted scaffolds (UCIM), (c) cell-seeded scaffolds in static culture (CIV) and (d) unseeded scaffolds in static culture (UCIV). The reduction in water proton transverse relaxation times and the concomitant increase in water proton magnetization transfer ratios for CIM and CIV scaffolds, compared to UCIV scaffolds, were consistent with the formation of a bone-like tissue within the polymer scaffold, which was confirmed by immunohistochemistry and fluorescence microscopy. However, the presence of angiogenic vessels and fibrotic adhesions around UCIM scaffolds can confound MRI findings of bone deposition. Consequently, to improve the specificity of the MRI technique for detecting mineralized deposits within explanted tissue engineered bone constructs, we introduce a novel contrast agent that uses alendronate to target a Food and Drug Administration-approved MRI contrast agent (Gd-DOTA) to bone mineral. Our contrast agent termed GdALN was used to uniquely identify mineralized deposits in representative samples from our four experimental groups. After GdALN treatment, both CIM and CIV scaffolds, containing mineralized deposits, showed marked signal enhancement on longitudinal relaxation time-weighted (T1W) images compared to UCIV scaffolds. Relative to UCIV scaffolds, some enhancement was observed in T1W images of GdALN-treated UCIM scaffolds, subjacent to the dark adhesions at the scaffold surface, possibly from dystrophic mineral formed in the fibrotic adhesions. Notably, residual dark areas on T1W images of CIM and UCIM scaffolds were attributable to blood inside infiltrating vessels. In summary, we present the efficacy of GdALN for sensitizing the MRI technique to the deposition of mineralized deposits in explanted polymeric scaffolds.


Subject(s)
Bone Substitutes/chemistry , Chorioallantoic Membrane/metabolism , Contrast Media/pharmacology , Magnetic Resonance Imaging/methods , Alendronate/pharmacology , Animals , Bone and Bones/pathology , Chickens , Fibrosis/pathology , Heterocyclic Compounds/pharmacology , Immunohistochemistry/methods , Microscopy, Fluorescence/methods , Organometallic Compounds/pharmacology , Osteoblasts/cytology , Ovum/pathology , Polymers/chemistry , Prostheses and Implants , Tissue Engineering/methods , Tissue Scaffolds
11.
J Mol Diagn ; 13(3): 282-8, 2011 May.
Article in English | MEDLINE | ID: mdl-21497290

ABSTRACT

Formalin-fixed, paraffin-embedded tissues generally provide low yields of extractable RNA that exhibit both covalent modification of nucleic acid bases and strand cleavage. This frustrates efforts to perform retrospective analyses of gene expression using archival tissue specimens. A variety of conditions have been reported to demodify formaldehyde-fixed RNA in different model systems. We studied the reversal of formaldehyde fixation of RNA using a 50 base RNA oligonucleotide and total cellular RNA. Formaldehyde-adducted, native, and hydrolyzed RNA species were identified by their bioanalyzer electrophoretic migration patterns and RT-quantitative PCR. Demodification conditions included temperature, time, buffer, and pH. The reversal of formaldehyde-fixed RNA to native species without apparent RNA hydrolysis was most successfully performed in dilute Tris, phosphate, or similar buffers (pH 8) at 70°C for 30 minutes. Amines were not required for efficient formaldehyde demodification. Formaldehyde-fixed RNA was more labile than native RNA to treatment with heat and buffer, suggesting that antigen retrieval methods for proteins may impede RNA hybridization or RNA extraction. Taken together, the data indicate that reliable conditions may be used to remove formaldehyde adducts from RNA to improve the quality of RNA available for molecular studies.


Subject(s)
Fixatives/pharmacology , Formaldehyde/chemistry , Formaldehyde/pharmacology , RNA/drug effects , Tissue Fixation , Buffers , HeLa Cells , Histological Techniques , Humans , Hydrogen-Ion Concentration , Temperature , Time Factors
12.
Methods Mol Biol ; 724: 1-21, 2011.
Article in English | MEDLINE | ID: mdl-21370003

ABSTRACT

Formalin-fixed, paraffin-embedded (FFPE) archival tissues and their associated diagnostic records represent an invaluable source of information on diseases where the patient outcomes are already known. Older archives contain many unique FFPE tissue specimens that would be impossible to replicate today due to changes in medical practice and technology. Unfortunately, there is no single regulatory or bioethical standard that covers research with FFPE tissue specimens. This makes it difficult for researchers to prepare protocols involving FFPE tissues and equally difficult for Institutional Review Boards to evaluate them. In this review, focused on US regulatory policy, the application of the Common Rule and the Privacy Rule of the Health Insurance Portability and Accountability Act to research involving FFPE tissue specimens will be discussed. It will be shown that the difficulty in applying regulatory and ethical standards to FFPE tissues results not from the tissues themselves, but from the personally identifiable health information associated with the tissue specimens.


Subject(s)
Ethics, Research , Formaldehyde/chemistry , Paraffin Embedding/ethics , Paraffin Embedding/methods , Research/legislation & jurisprudence , Tissue Fixation/ethics , Tissue Fixation/methods , Informed Consent
13.
Methods Mol Biol ; 724: 23-35, 2011.
Article in English | MEDLINE | ID: mdl-21370004

ABSTRACT

Tissue microarrays (TMAs) are produced by taking small punches from a series of paraffin-embedded (donor) tissue blocks and transferring these tissue cores into a positionally encoded array in a recipient paraffin block. Though TMAs are not used for clinical diagnosis, they have several advantages over using conventional whole histological sections for research. Tissue from multiple patients or blocks can be examined on the same slide, and only a very small amount of reagent is required to stain or label an entire array. Multiple sections (100-300) can be cut from a single array block, allowing for hundreds of analyses per microarray. These advantages allow the use of TMAs in high-throughput procedures, such as screening antibodies for diagnostics and validating prognostic markers that are impractical using conventional whole tissue sections. TMAs can be used for immunohistochemistry, immunofluorescence, in situ hybridization, and conventional histochemical staining. Finally, several tissue cores may be taken without -consuming the tissue block, allowing the donor block to be returned to its archive for any additional studies.


Subject(s)
Tissue Array Analysis/methods , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Eosine Yellowish-(YS)/metabolism , Female , Hematoxylin/metabolism , Humans , In Situ Hybridization, Fluorescence , Receptor, ErbB-2/metabolism , Staining and Labeling
14.
Methods Mol Biol ; 724: 281-95, 2011.
Article in English | MEDLINE | ID: mdl-21370020

ABSTRACT

Formalin-fixed, paraffin-embedded (FFPE) tissue archives and their associated diagnostic records represent an invaluable source of proteomic information on diseases where the patient outcomes are already known. Over the last few years, advances in methodology have made it possible to recover peptides from FFPE tissues that yield a reasonable representation of the proteins recovered from identical fresh or frozen specimens. These new methods, based largely upon heat-induced antigen retrieval techniques borrowed from immunohistochemistry, have developed sufficiently to allow at least a qualitative analysis of the proteome of FFPE archival tissues. This chapter describes the approaches for performing proteomic analysis on FFPE tissues by liquid chromatography and mass spectrometry.


Subject(s)
Formaldehyde/chemistry , Mass Spectrometry/methods , Paraffin Embedding/methods , Proteins/chemistry , Proteins/metabolism , Tissue Fixation/methods , Chromatography, Liquid , Databases, Protein , Electrophoresis, Polyacrylamide Gel , Humans , Proteins/isolation & purification , Proteomics , Solutions , Statistics as Topic , Trypsin/metabolism
15.
J Histochem Cytochem ; 59(4): 366-81, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21411808

ABSTRACT

Antigen retrieval (AR), in which formalin-fixed paraffin-embedded tissue sections are briefly heated in buffers at high temperature, often greatly improves immunohistochemical staining. An important unresolved question regarding AR is how formalin treatment affects the conformation of protein epitopes and how heating unmasks these epitopes for subsequent antibody binding. The objective of the current study was to use model proteins to determine the effect of formalin treatment on protein conformation and thermal stability in relation to the mechanism of AR. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to identify the presence of protein formaldehyde cross-links, and circular dichroism spectropolarimetry was used to determine the effect of formalin treatment and high-temperature incubation on the secondary and tertiary structure of the model proteins. Results revealed that for some proteins, formalin treatment left the native protein conformation unaltered, whereas for others, formalin denatured tertiary structure, yielding a molten globule protein. In either case, heating to temperatures used in AR methods led to irreversible protein unfolding, which supports a linear epitope model of recovered protein immunoreactivity. Consequently, the core mechanism of AR likely centers on the restoration of normal protein chemical composition coupled with improved accessibility to linear epitopes through protein unfolding.


Subject(s)
Antigens/chemistry , Formaldehyde , Protein Unfolding , Proteins/chemistry , Circular Dichroism , Cross-Linking Reagents , Electrophoresis, Polyacrylamide Gel , Epitopes , Fixatives , Hot Temperature , Paraffin Embedding , Protein Denaturation , Protein Structure, Secondary , Protein Structure, Tertiary
16.
Cancer Epidemiol ; 35(5): 475-9, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21292584

ABSTRACT

BACKGROUND: Our previous studies revealed that leukocyte infiltration into aged or injured myoepithelial cell layers is a key trigger for breast tumor invasion and metastasis. Our current study further assessed the possibility that leukocyte aggregates may harbor detached individual tumor cell or clusters of tumor cells. MATERIALS AND METHODS: Tissue sections from patients with pregnancy-associated breast cancer (PABC) and controls were subjected to morphological and immunohistochemical assessment with a panel of leukocyte and tumor cell related markers. RESULTS: A total of 63 leukocyte aggregates were detected in the 20 PABC cases studied. Of these, 55 (87%) were distributed within normal or hyperplastic lobules adjacent to invasive lesions. Over 70% of these leukocyte aggregates harbored detached individual tumor cell or cell clusters with malignant properties, including strong p53 positivity, elevated proliferation, reduced cell surface adhesion molecules, and cytological resemblance to adjacent invasive cancer cells. A significant number of these tumor cells or condensed chromosomes of mitotic tumor cells were observed to conjoin with the plasma membrane of leukocytes. Similar alterations were seen in leukocyte aggregates within the inter-lobular space and in non-PABC with a lower frequency. CONCLUSIONS: These findings suggest that leukocyte infiltration may trigger dissemination of tumor cells from their primary site, and that leukocyte aggregates may serve as a reservoir for disseminated tumor cells that may be physically dragged to distant sites by leukocytes during their migration.


Subject(s)
Breast Neoplasms/pathology , Epithelial Cells/pathology , Leukocytes/pathology , Muscle Cells/pathology , Myoepithelioma/pathology , Neoplasm Seeding , Pregnancy Complications, Neoplastic/pathology , Adult , Biomarkers, Tumor/metabolism , Breast/metabolism , Breast/pathology , Breast Neoplasms/metabolism , Case-Control Studies , Epithelial Cells/metabolism , Female , Humans , Immunoenzyme Techniques , Leukocytes/metabolism , Muscle Cells/metabolism , Myoepithelioma/metabolism , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Staging , Pregnancy , Pregnancy Complications, Neoplastic/metabolism , Prognosis , Young Adult
17.
PLoS One ; 5(12): e14253, 2010 Dec 08.
Article in English | MEDLINE | ID: mdl-21170380

ABSTRACT

BACKGROUND: Proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing. PRINCIPAL FINDINGS: In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%). Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis) of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates. CONCLUSIONS: These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form suitable for proteomic analysis.


Subject(s)
Paraffin Embedding/methods , Paraffin/chemistry , Proteins/chemistry , Tissue Fixation/methods , Animals , Chickens , Cross-Linking Reagents/chemistry , Egg White/chemistry , Electrophoresis, Gel, Two-Dimensional , Fixatives/chemistry , Formaldehyde/chemistry , Liver/metabolism , Mass Spectrometry/methods , Mice , Muramidase/chemistry
18.
J Cancer ; 1: 178-83, 2010 Oct 12.
Article in English | MEDLINE | ID: mdl-20975848

ABSTRACT

Formaldehyde fixation and paraffin-embedding remains the most widely used technique for processing cancer tissue specimens for pathologic examination, the study of tissue morphology, and archival preservation. However, formaldehyde penetration and fixation is a slow process, requiring a minimum of 15 hr for routine processing of pathology samples. Routinely fixed samples often have a well-fixed outer rim, with a poorly-fixed inner core of tissue. In this study, we show that the application of elevated pressure up to 15,000 psi improves the rate of formaldehyde fixation by approximately 5 to 7-fold while preserving the tissue morphology of porcine liver. The tissue also exhibited much more uniform formaldehyde penetration after 30-60 min incubation under elevated pressure than samples fixed for the same length of time at atmospheric pressure.

19.
J Clin Microbiol ; 48(12): 4620-2, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20962142

ABSTRACT

Practical detection of cholera toxin (CT) by a liposome PCR (LPCR) immunoassay was compared to that of an established V. cholerae enterotoxin and Escherichia coli heat-labile enterotoxin reversed passive latex agglutination (VET-RPLA) assay. LPCR detected CT in the range of 10 pg/ml to 100 ng/ml in simulated feces and environmental water. Detection by VET-RPLA required at least 4 to 19 ng/ml CT.


Subject(s)
Cholera Toxin/analysis , Feces/chemistry , Liposomes , Polymerase Chain Reaction/methods , Water/analysis , Bacterial Toxins/analysis , Enterotoxins/analysis , Escherichia coli Proteins/analysis , Humans , Immunoassay/methods , Latex Fixation Tests , Sensitivity and Specificity
20.
J Cancer ; 1: 6-13, 2010 Jun 01.
Article in English | MEDLINE | ID: mdl-20842218

ABSTRACT

Human breast cancer represents a group of highly heterogeneous lesions consisting of about 20 morphologically distinct subtypes with substantially different molecular and/or biochemical signatures, clinical courses, and prognoses. This study analyzed the possible correlation between the morphological presentations of breast cancer and two hypothesized models of carcinogenesis, in order to identify the intrinsic mechanism(s) and clinical implications of breast cancer heterogeneity.

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