Science.gov

Sample records for addition tissue samples

  1. SEM investigation of heart tissue samples

    NASA Astrophysics Data System (ADS)

    Saunders, R.; Amoroso, M.

    2010-07-01

    We used the scanning electron microscope to examine the cardiac tissue of a cow (Bos taurus), a pig (Sus scrofa), and a human (Homo sapiens). 1mm3 blocks of left ventricular tissue were prepared for SEM scanning by fixing in 96% ethanol followed by critical point drying (cryofixation), then sputter-coating with gold. The typical ridged structure of the myofibrils was observed for all the species. In addition crystal like structures were found in one of the samples of the heart tissue of the pig. These structures were investigated further using an EDVAC x-ray analysis attachment to the SEM. Elemental x-ray analysis showed highest peaks occurred for gold, followed by carbon, oxygen, magnesium and potassium. As the samples were coated with gold for conductivity, this highest peak is expected. Much lower peaks at carbon, oxygen, magnesium and potassium suggest that a cystallized salt such as a carbonate was present in the tissue before sacrifice.

  2. Combined additive manufacturing approaches in tissue engineering.

    PubMed

    Giannitelli, S M; Mozetic, P; Trombetta, M; Rainer, A

    2015-09-01

    Advances introduced by additive manufacturing (AM) have significantly improved the control over the microarchitecture of scaffolds for tissue engineering. This has led to the flourishing of research works addressing the optimization of AM scaffolds microarchitecture to optimally trade-off between conflicting requirements (e.g. mechanical stiffness and porosity level). A fascinating trend concerns the integration of AM with other scaffold fabrication methods (i.e. "combined" AM), leading to hybrid architectures with complementary structural features. Although this innovative approach is still at its beginning, significant results have been achieved in terms of improved biological response to the scaffold, especially targeting the regeneration of complex tissues. This review paper reports the state of the art in the field of combined AM, posing the accent on recent trends, challenges, and future perspectives.

  3. Additive Manufacturing of Biomaterials, Tissues, and Organs.

    PubMed

    Zadpoor, Amir A; Malda, Jos

    2017-01-01

    The introduction of additive manufacturing (AM), often referred to as three-dimensional (3D) printing, has initiated what some believe to be a manufacturing revolution, and has expedited the development of the field of biofabrication. Moreover, recent advances in AM have facilitated further development of patient-specific healthcare solutions. Customization of many healthcare products and services, such as implants, drug delivery devices, medical instruments, prosthetics, and in vitro models, would have been extremely challenging-if not impossible-without AM technologies. The current special issue of the Annals of Biomedical Engineering presents the latest trends in application of AM techniques to healthcare-related areas of research. As a prelude to this special issue, we review here the most important areas of biomedical research and clinical practice that have benefited from recent developments in additive manufacturing techniques. This editorial, therefore, aims to sketch the research landscape within which the other contributions of the special issue can be better understood and positioned. In what follows, we briefly review the application of additive manufacturing techniques in studies addressing biomaterials, (re)generation of tissues and organs, disease models, drug delivery systems, implants, medical instruments, prosthetics, orthotics, and AM objects used for medical visualization and communication.

  4. Measuring the elastic modulus of small tissue samples.

    PubMed

    Erkamp, R Q; Wiggins, P; Skovoroda, A R; Emelianov, S Y; O'Donnell, M

    1998-01-01

    Independent measurements of the elastic modulus (Young's modulus) of tissue are necessary step in turning elasticity imaging into a clinical tool. A system capable of measuring the elastic modulus of small tissue samples was developed. The system tolerates the constraints of biological tissue, such as limited sample size (< or = 1.5 cm3) and imperfections in sample geometry. A known deformation is applied to the tissue sample while simultaneously measuring the resulting force. These measurements are then converted to an elastic modulus, where the conversion uses prior calibration of the system with plastisol samples of known Young's modulus. Accurate measurements have been obtained from 10 to 80 kPa, covering a wide range of tissue modulus values. In addition, the performance of the system was further investigated using finite element analysis. Finally, preliminary elasticity measurements on canine kidney samples are presented and discussed.

  5. Tissue Sampling Guides for Porcine Biomedical Models.

    PubMed

    Albl, Barbara; Haesner, Serena; Braun-Reichhart, Christina; Streckel, Elisabeth; Renner, Simone; Seeliger, Frank; Wolf, Eckhard; Wanke, Rüdiger; Blutke, Andreas

    2016-04-01

    This article provides guidelines for organ and tissue sampling adapted to porcine animal models in translational medical research. Detailed protocols for the determination of sampling locations and numbers as well as recommendations on the orientation, size, and trimming direction of samples from ∼50 different porcine organs and tissues are provided in the Supplementary Material. The proposed sampling protocols include the generation of samples suitable for subsequent qualitative and quantitative analyses, including cryohistology, paraffin, and plastic histology; immunohistochemistry;in situhybridization; electron microscopy; and quantitative stereology as well as molecular analyses of DNA, RNA, proteins, metabolites, and electrolytes. With regard to the planned extent of sampling efforts, time, and personnel expenses, and dependent upon the scheduled analyses, different protocols are provided. These protocols are adjusted for (I) routine screenings, as used in general toxicity studies or in analyses of gene expression patterns or histopathological organ alterations, (II) advanced analyses of single organs/tissues, and (III) large-scale sampling procedures to be applied in biobank projects. Providing a robust reference for studies of porcine models, the described protocols will ensure the efficiency of sampling, the systematic recovery of high-quality samples representing the entire organ or tissue as well as the intra-/interstudy comparability and reproducibility of results.

  6. Lidocaine Concentration in Oral Tissue by the Addition of Epinephrine.

    PubMed

    Tanaka, Eri; Yoshida, Kenji; Kawaai, Hiroyoshi; Yamazaki, Shinya

    2016-01-01

    The vasoconstrictive effect due to the addition of epinephrine to local anesthetic has been clearly shown by measuring blood-flow volume or blood anesthetic concentration in oral mucosal tissue. However, there are no reports on the measurement of anesthetic concentration using samples directly taken from the jawbone and oral mucosal tissue. Consequently, in this study, the effect of lidocaine concentration in the jawbone and oral mucosal tissue by the addition of epinephrine to the local anesthetic lidocaine was considered by quantitatively measuring lidocaine concentration within the tissue. Japanese white male rabbits (n = 96) were used as test animals. General anesthesia was induced by sevoflurane and oxygen, and then cannulation to the femoral artery was performed while arterial pressure was constantly recorded. Infiltration anesthesia was achieved by 0.5 mL of 2% lidocaine containing 1 : 80,000 epinephrine in the upper jawbone (E(+)) and 0.5 mL of 2% of epinephrine additive-free lidocaine (E(0)) under the periosteum. At specified time increments (10, 20, 30, 40, 50, and 60 minutes), samples from the jawbone, oral mucosa, and blood were collected, and lidocaine concentration was directly measured by high-performance liquid chromatography. No significant differences in the change in blood pressure were observed either in E(+) or E(0). In both E(+) and E(0) groups, the serum lidocaine concentration peaked 10 minutes after local anesthesia and decreased thereafter. At all time increments, serum lidocaine concentration in E(+) was significantly lower than that in E(0). There were no significant differences in measured lidocaine concentration between jawbone and mucosa within either the E(+) or the E(0) groups at all time points, although the E(0) group had significantly lower jawbone and mucosa concentrations than the E(+) group at all time points when comparing the 2 groups to each other. Addition of epinephrine to the local anesthetic inhibited systemic

  7. Improved selenium recovery from tissue with modified sample decomposition

    USGS Publications Warehouse

    Brumbaugh, W. G.; Walther, M.J.

    1991-01-01

    The present paper describes a simple modification of a recently reported decomposition method for determination of selenium in biological tissue by hydride generation atomic absorption. The modified method yielded slightly higher selenium recoveries (3-4%) for selected reference tissues and fish tissue spiked with selenomethionine. Radiotracer experiments indicated that the addition of a small volume of hydrochloric acid to the wet digestate mixture reduced slight losses of selenium as the sample initially went to dryness before ashing. With the modified method, selenium spiked as selenomethionine behaved more like the selenium in reference tissues than did the inorganic spike forms when this digestion modification was used.

  8. Lidocaine Concentration in Oral Tissue by the Addition of Epinephrine

    PubMed Central

    Tanaka, Eri; Yoshida, Kenji; Kawaai, Hiroyoshi; Yamazaki, Shinya

    2016-01-01

    The vasoconstrictive effect due to the addition of epinephrine to local anesthetic has been clearly shown by measuring blood-flow volume or blood anesthetic concentration in oral mucosal tissue. However, there are no reports on the measurement of anesthetic concentration using samples directly taken from the jawbone and oral mucosal tissue. Consequently, in this study, the effect of lidocaine concentration in the jawbone and oral mucosal tissue by the addition of epinephrine to the local anesthetic lidocaine was considered by quantitatively measuring lidocaine concentration within the tissue. Japanese white male rabbits (n = 96) were used as test animals. General anesthesia was induced by sevoflurane and oxygen, and then cannulation to the femoral artery was performed while arterial pressure was constantly recorded. Infiltration anesthesia was achieved by 0.5 mL of 2% lidocaine containing 1 : 80,000 epinephrine in the upper jawbone (E+) and 0.5 mL of 2% of epinephrine additive–free lidocaine (E0) under the periosteum. At specified time increments (10, 20, 30, 40, 50, and 60 minutes), samples from the jawbone, oral mucosa, and blood were collected, and lidocaine concentration was directly measured by high-performance liquid chromatography. No significant differences in the change in blood pressure were observed either in E+ or E0. In both E+ and E0 groups, the serum lidocaine concentration peaked 10 minutes after local anesthesia and decreased thereafter. At all time increments, serum lidocaine concentration in E+ was significantly lower than that in E0. There were no significant differences in measured lidocaine concentration between jawbone and mucosa within either the E+ or the E0 groups at all time points, although the E0 group had significantly lower jawbone and mucosa concentrations than the E+ group at all time points when comparing the 2 groups to each other. Addition of epinephrine to the local anesthetic inhibited systemic absorption of local

  9. 7 CFR 27.25 - Additional samples of cotton; drawing.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Additional samples of cotton; drawing. 27.25 Section... CONTAINER REGULATIONS COTTON CLASSIFICATION UNDER COTTON FUTURES LEGISLATION Regulations Inspection and Samples § 27.25 Additional samples of cotton; drawing. In addition to the samples hereinbefore...

  10. 7 CFR 27.25 - Additional samples of cotton; drawing.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Additional samples of cotton; drawing. 27.25 Section... CONTAINER REGULATIONS COTTON CLASSIFICATION UNDER COTTON FUTURES LEGISLATION Regulations Inspection and Samples § 27.25 Additional samples of cotton; drawing. In addition to the samples hereinbefore...

  11. 7 CFR 27.25 - Additional samples of cotton; drawing.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Additional samples of cotton; drawing. 27.25 Section... CONTAINER REGULATIONS COTTON CLASSIFICATION UNDER COTTON FUTURES LEGISLATION Regulations Inspection and Samples § 27.25 Additional samples of cotton; drawing. In addition to the samples hereinbefore...

  12. 7 CFR 27.25 - Additional samples of cotton; drawing.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Additional samples of cotton; drawing. 27.25 Section... CONTAINER REGULATIONS COTTON CLASSIFICATION UNDER COTTON FUTURES LEGISLATION Regulations Inspection and Samples § 27.25 Additional samples of cotton; drawing. In addition to the samples hereinbefore...

  13. 7 CFR 27.25 - Additional samples of cotton; drawing.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Additional samples of cotton; drawing. 27.25 Section... CONTAINER REGULATIONS COTTON CLASSIFICATION UNDER COTTON FUTURES LEGISLATION Regulations Inspection and Samples § 27.25 Additional samples of cotton; drawing. In addition to the samples hereinbefore...

  14. Additive Biomanufacturing: An Advanced Approach for Periodontal Tissue Regeneration.

    PubMed

    Carter, Sarah-Sophia D; Costa, Pedro F; Vaquette, Cedryck; Ivanovski, Saso; Hutmacher, Dietmar W; Malda, Jos

    2017-01-01

    Periodontitis is defined as a chronic inflammatory condition, characterized by destruction of the periodontium, composed of hard (i.e. alveolar bone and cementum) and soft tissues (i.e. gingiva and periodontal ligament) surrounding and supporting the teeth. In severe cases, reduced periodontal support can lead to tooth loss, which requires tissue augmentation or procedures that initiate a repair, yet ideally a regenerative response. However, mimicking the three-dimensional complexity and functional integration of the different tissue components via scaffold- and/or matrix-based guided tissue engineering represents a great challenge. Additive biomanufacturing, a manufacturing method in which objects are designed and fabricated in a layer-by-layer manner, has allowed a paradigm shift in the current manufacturing of medical devices and implants. This shift from design-to-manufacture to manufacture-to-design, seen from a translational research point of view, provides the biomedical engineering and periodontology communities a technology with the potential to achieve tissue regeneration instead of repair. In this review, the focus is put on additively biomanufactured scaffolds for periodontal applications. Besides a general overview of the concept of additive biomanufacturing within this field, different developed scaffold designs are described. To conclude, future directions regarding advanced biomaterials and additive biomanufacturing technologies for applications in regenerative periodontology are highlighted.

  15. 21 CFR 71.4 - Samples; additional information.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 1 2013-04-01 2013-04-01 false Samples; additional information. 71.4 Section 71.4 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL COLOR... samples of the color additive, articles used as components thereof, or of the food, drug, or cosmetic...

  16. 21 CFR 71.4 - Samples; additional information.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 1 2012-04-01 2012-04-01 false Samples; additional information. 71.4 Section 71.4 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL COLOR... samples of the color additive, articles used as components thereof, or of the food, drug, or cosmetic...

  17. Additive manufacturing techniques for the production of tissue engineering constructs.

    PubMed

    Mota, Carlos; Puppi, Dario; Chiellini, Federica; Chiellini, Emo

    2015-03-01

    'Additive manufacturing' (AM) refers to a class of manufacturing processes based on the building of a solid object from three-dimensional (3D) model data by joining materials, usually layer upon layer. Among the vast array of techniques developed for the production of tissue-engineering (TE) scaffolds, AM techniques are gaining great interest for their suitability in achieving complex shapes and microstructures with a high degree of automation, good accuracy and reproducibility. In addition, the possibility of rapidly producing tissue-engineered constructs meeting patient's specific requirements, in terms of tissue defect size and geometry as well as autologous biological features, makes them a powerful way of enhancing clinical routine procedures. This paper gives an extensive overview of different AM techniques classes (i.e. stereolithography, selective laser sintering, 3D printing, melt-extrusion-based techniques, solution/slurry extrusion-based techniques, and tissue and organ printing) employed for the development of tissue-engineered constructs made of different materials (i.e. polymeric, ceramic and composite, alone or in combination with bioactive agents), by highlighting their principles and technological solutions.

  18. Additive Manufacturing of Vascular Grafts and Vascularized Tissue Constructs.

    PubMed

    Elomaa, Laura; Yang, Yunzhi Peter

    2017-01-10

    There is a great need for engineered vascular grafts among patients with cardiovascular diseases who are in need of bypass therapy and lack autologous healthy blood vessels. In addition, because of the severe worldwide shortage of organ donors, there is an increasing need for engineered vascularized tissue constructs as an alternative to organ transplants. Additive manufacturing (AM) offers great advantages and flexibility of fabrication of cell-laden, multimaterial, and anatomically shaped vascular grafts and vascularized tissue constructs. Various inkjet-, extrusion-, and photocrosslinking-based AM techniques have been applied to the fabrication of both self-standing vascular grafts and porous, vascularized tissue constructs. This review discusses the state-of-the-art research on the use of AM for vascular applications and the key criteria for biomaterials in the AM of both acellular and cellular constructs. We envision that new smart printing materials that can adapt to their environment and encourage rapid endothelialization and remodeling will be the key factor in the future for the successful AM of personalized and dynamic vascular tissue applications.

  19. [Analysis of human tissue samples for volatile fire accelerants].

    PubMed

    Treibs, Rudolf

    2014-01-01

    In police investigations of fires, the cause of a fire and the fire debris analysis regarding traces of fire accelerants are important aspects for forensic scientists. Established analytical procedures were recently applied to the remains of fire victims. When examining lung tissue samples, vapors inhaled from volatile ignitable liquids could be identified and differentiated from products of pyrolysis caused by the fire. In addition to the medico-legal results this evidence allowed to draw conclusions as to whether the fire victim was still alive when the fire started.

  20. Variation in glycogen concentrations within mantle and foot tissue in Amblema plicata plicata: Implications for tissue biopsy sampling

    USGS Publications Warehouse

    Naimo, T.J.; Monroe, E.M.

    1999-01-01

    With the development of techniques to non-lethally biopsy tissue from unionids, a new method is available to measure changes in biochemical, contaminant, and genetic constituents in this imperiled faunal group. However, before its widespread application, information on the variability of biochemical components within and among tissues needs to be evaluated. We measured glycogen concentrations in foot and mantle tissue in Amblema plicata plicata (Say, 1817) to determine if glycogen was evenly distributed within and between tissues and to determine which tissue might be more responsive to the stress associated with relocating mussels. Glycogen was measured in two groups of mussels: those sampled from their native environment (undisturbed mussels) and quickly frozen for analysis and those relocated into an artificial pond (relocated mussels) for 24 months before analysis. In both undisturbed and relocated mussels, glycogen concentrations were evenly distributed within foot, but not within mantle tissue. In mantle tissue, concentrations of glycogen varied about 2-fold among sections. In addition, glycogen varied significantly between tissues in undisturbed mussels, but not in relocated mussels. Twenty-four months after relocation, glycogen concentrations had declined by 80% in mantle tissue and by 56% in foot tissue relative to the undisturbed mussels. These data indicate that representative biopsy samples can be obtained from foot tissue, but not mantle tissue. We hypothesize that mantle tissue could be more responsive to the stress of relocation due to its high metabolic activity associated with shell formation.

  1. 76 FR 82308 - Guidance for Industry: Current Good Tissue Practice and Additional Requirements for Manufacturers...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-30

    ... Additional Requirements for Manufacturers of Human Cells, Tissues, and Cellular and Tissue-Based Products... Tissue Practice (CGTP) and Additional Requirements for Manufacturers of Human Cells, Tissues, and... for Manufacturers of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps)''...

  2. Leaf tissue sampling and DNA extraction protocols.

    PubMed

    Semagn, Kassa

    2014-01-01

    Taxonomists must be familiar with a number of issues in collecting and transporting samples using freezing methods (liquid nitrogen and dry ice), desiccants (silica gel and blotter paper), and preservatives (CTAB, ethanol, and isopropanol), with each method having its own merits and limitations. For most molecular studies, a reasonably good quality and quantity of DNA is required, which can only be obtained using standard DNA extraction protocols. There are many DNA extraction protocols that vary from simple and quick ones that yield low-quality DNA but good enough for routine analyses to the laborious and time-consuming standard methods that usually produce high quality and quantities of DNA. The protocol to be chosen will depend on the quality and quantity of DNA needed, the nature of samples, and the presence of natural substances that may interfere with the extraction and subsequent analysis. The protocol described in this chapter has been tested for extracting DNA from eight species and provided very good quality and quantity of DNA for different applications, including those genotyping methods that use restriction enzymes.

  3. An effective plasma membrane proteomics approach for small tissue samples

    PubMed Central

    Smolders, Katrien; Lombaert, Nathalie; Valkenborg, Dirk; Baggerman, Geert; Arckens, Lutgarde

    2015-01-01

    Advancing the quest for new drug targets demands the development of innovative plasma membrane proteome research strategies applicable to small, functionally defined tissue samples. Biotinylation of acute tissue slices and streptavidin pull-down followed by shotgun proteomics allowed the selective extraction and identification of >1,600 proteins of which >60% are associated with the plasma membrane, including (G-protein coupled) receptors, ion channels and transporters, and this from mm3-scale tissue. PMID:26047021

  4. Measuring the elastic modulus of ex vivo small tissue samples

    NASA Astrophysics Data System (ADS)

    Samani, Abbas; Bishop, Jonathan; Luginbuhl, Chris; Plewes, Donald B.

    2003-07-01

    Over the past decade, several methods have been proposed to image tissue elasticity based on imaging methods collectively called elastography. While progress in developing these systems has been rapid, the basic understanding of tissue properties to interpret elastography images is generally lacking. To address this limitation, we developed a system to measure the Young's modulus of small soft tissue specimens. This system was designed to accommodate biological soft tissue constraints such as sample size, geometry imperfection and heterogeneity. The measurement technique consists of indenting an unconfined small block of tissue while measuring the resulting force. We show that the measured force-displacement slope of such a geometry can be transformed to the tissue Young's modulus via a conversion factor related to the sample's geometry and boundary conditions using finite element analysis. We also demonstrate another measurement technique for tissue elasticity based on quasi-static magnetic resonance elastography in which a tissue specimen encased in a gelatine-agarose block undergoes cyclical compression with resulting displacements measured using a phase contrast MRI technique. The tissue Young's modulus is then reconstructed from the measured displacements using an inversion technique. Finally, preliminary elasticity measurement results of various breast tissues are presented and discussed.

  5. Long-term room temperature preservation of corpse soft tissue: an approach for tissue sample storage

    PubMed Central

    2011-01-01

    Background Disaster victim identification (DVI) represents one of the most difficult challenges in forensic sciences, and subsequent DNA typing is essential. Collected samples for DNA-based human identification are usually stored at low temperature to halt the degradation processes of human remains. We have developed a simple and reliable procedure for soft tissue storage and preservation for DNA extraction. It ensures high quality DNA suitable for PCR-based DNA typing after at least 1 year of room temperature storage. Methods Fragments of human psoas muscle were exposed to three different environmental conditions for diverse time periods at room temperature. Storage conditions included: (a) a preserving medium consisting of solid sodium chloride (salt), (b) no additional substances and (c) garden soil. DNA was extracted with proteinase K/SDS followed by organic solvent treatment and concentration by centrifugal filter devices. Quantification was carried out by real-time PCR using commercial kits. Short tandem repeat (STR) typing profiles were analysed with 'expert software'. Results DNA quantities recovered from samples stored in salt were similar up to the complete storage time and underscored the effectiveness of the preservation method. It was possible to reliably and accurately type different genetic systems including autosomal STRs and mitochondrial and Y-chromosome haplogroups. Autosomal STR typing quality was evaluated by expert software, denoting high quality profiles from DNA samples obtained from corpse tissue stored in salt for up to 365 days. Conclusions The procedure proposed herein is a cost efficient alternative for storage of human remains in challenging environmental areas, such as mass disaster locations, mass graves and exhumations. This technique should be considered as an additional method for sample storage when preservation of DNA integrity is required for PCR-based DNA typing. PMID:21846338

  6. Specimen Sample Preservation for Cell and Tissue Cultures

    NASA Technical Reports Server (NTRS)

    Meeker, Gabrielle; Ronzana, Karolyn; Schibner, Karen; Evans, Robert

    1996-01-01

    The era of the International Space Station with its longer duration missions will pose unique challenges to microgravity life sciences research. The Space Station Biological Research Project (SSBRP) is responsible for addressing these challenges and defining the science requirements necessary to conduct life science research on-board the International Space Station. Space Station will support a wide range of cell and tissue culture experiments for durations of 1 to 30 days. Space Shuttle flights to bring experimental samples back to Earth for analyses will only occur every 90 days. Therefore, samples may have to be retained for periods up to 60 days. This presents a new challenge in fresh specimen sample storage for cell biology. Fresh specimen samples are defined as samples that are preserved by means other than fixation and cryopreservation. The challenge of long-term storage of fresh specimen samples includes the need to suspend or inhibit proliferation and metabolism pending return to Earth-based laboratories. With this challenge being unique to space research, there have not been any ground based studies performed to address this issue. It was decided hy SSBRP that experiment support studies to address the following issues were needed: Fixative Solution Management; Media Storage Conditions; Fresh Specimen Sample Storage of Mammalian Cell/Tissue Cultures; Fresh Specimen Sample Storage of Plant Cell/Tissue Cultures; Fresh Specimen Sample Storage of Aquatic Cell/Tissue Cultures; and Fresh Specimen Sample Storage of Microbial Cell/Tissue Cultures. The objective of these studies was to derive a set of conditions and recommendations that can be used in a long duration microgravity environment such as Space Station that will permit extended storage of cell and tissue culture specimens in a state consistent with zero or minimal growth, while at the same time maintaining their stability and viability.

  7. Analysis of chemical components from plant tissue samples

    NASA Technical Reports Server (NTRS)

    Laseter, J. L.

    1972-01-01

    Information is given on the type and concentration of sterols, free fatty acids, and total fatty acids in plant tissue samples. All samples were analyzed by gas chromatography and then by gas chromatography-mass spectrometry combination. In each case the mass spectral data was accumulated as a computer printout and plot. Typical gas chromatograms are included as well as tables describing test results.

  8. Multivariate classification of infrared spectra of cell and tissue samples

    DOEpatents

    Haaland, David M.; Jones, Howland D. T.; Thomas, Edward V.

    1997-01-01

    Multivariate classification techniques are applied to spectra from cell and tissue samples irradiated with infrared radiation to determine if the samples are normal or abnormal (cancerous). Mid and near infrared radiation can be used for in vivo and in vitro classifications using at least different wavelengths.

  9. Additional sampling directions improve detection range of wireless radiofrequency probes

    PubMed Central

    Mada, Marius; Carpenter, T. Adrian; Sawiak, Stephen J.; Williams, Guy B.

    2015-01-01

    Purpose While MRI is enhancing our knowledge about the structure and function of the human brain, subject motion remains a problem in many clinical applications. Recently, the use of wireless radiofrequency markers with three one‐dimensional (1D) navigators for prospective correction was demonstrated. This method is restricted in the range of motion that can be corrected, however, because of limited information in the 1D readouts. Methods Here, the limitation of techniques for disambiguating marker locations was investigated. It was shown that including more sampling directions extends the tracking range for head rotations. The efficiency of trading readout resolution for speed was explored. Results Tracking of head rotations was demonstrated from −19.2 to 34.4°, −2.7 to 10.0°, and −60.9 to 70.9° in the x‐, y‐, and z‐directions, respectively. In the presence of excessive head motion, the deviation of marker estimates from SPM8 was reduced by 17.1% over existing three‐projection methods. This was achieved by using an additional seven directions, extending the time needed for readouts by a factor of 3.3. Much of this increase may be circumvented by reducing resolution, without compromising accuracy. Conclusion Including additional sampling directions extends the range in which markers can be used, for patients who move a lot. Magn Reson Med 76:913–918, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. PMID:26418189

  10. Surrogate matrix: opportunities and challenges for tissue sample analysis.

    PubMed

    Ho, Stacy; Gao, Hong

    2015-09-23

    Often there is limited availability of matching tissue matrix and/or the analyte may occur endogenously in the target tissue. Surrogate matrix provides an option for quantitation of drug, metabolite(s) and biomarker(s) in these circumstances. However, the use of a surrogate matrix also presents challenges. This paper summarizes and discusses the challenges of selecting a proper surrogate, validating the suitability of the surrogate and establishing a surrogate tissue method using the fit-for-purpose approach. This paper also systematically reviews the current practices for evaluating key parameters of a surrogate tissue assay, including sensitivity, specificity, selectivity, interference, precision, accuracy, recovery, matrix effects and stability. Considerations and suggestions are provided for dealing with such challenges during method establishment and tissue sample analysis.

  11. Coaxial additive manufacture of biomaterial composite scaffolds for tissue engineering.

    PubMed

    Cornock, R; Beirne, S; Thompson, B; Wallace, G G

    2014-06-01

    An inherent difficulty associated with the application of suitable bioscaffolds for tissue engineering is the incorporation of adequate mechanical characteristics into the materials which recapitulate that of the native tissue, whilst maintaining cell proliferation and nutrient transfer qualities. Biomaterial composites fabricated using rapid prototyping techniques can potentially improve the functionality and patient-specific processing of tissue engineering scaffolds. In this work, a technique for the coaxial melt extrusion printing of core-shell scaffold structures was designed, implemented and assessed with respect to the repeatability, cell efficacy and scaffold porosity obtainable. Encapsulated alginate hydrogel/thermoplastic polycaprolactone (Alg-PCL) cofibre scaffolds were fabricated. Selective laser melting was used to produce a high resolution stainless steel 316 L coaxial extrusion nozzle, exhibiting diameters of 300 μm/900 μm for the inner and outer nozzles respectively. We present coaxial melt extrusion printed scaffolds of Alg-PCL cofibres with ~0.4 volume fraction alginate, with total fibre diameter as low as 600 μm and core material offset as low as 10% of the total diameter. Furthermore the tuneability of scaffold porosity, pore size and interconnectivity, as well as the preliminary inclusion, compatibility and survival of an L-929 mouse fibroblast cell-line within the scaffolds were explored. This preliminary cell work highlighted the need for optimal material selection and further design reiteration in future research.

  12. Translational research in pediatrics: tissue sampling and biobanking.

    PubMed

    Brisson, Alayne R; Matsui, Doreen; Rieder, Michael J; Fraser, Douglas D

    2012-01-01

    Translational research is expanding and has become a focus of National Research funding agencies, touted as the primary avenue to improve health care practice. The use of human tissues for research on disease etiology is a pillar of translational research, particularly with innovations in research technologies to investigate the building blocks of disease. In pediatrics, translational research using human tissues has been hindered by the many practical and ethical considerations associated with tissue procurement from children and also by a limited population base for study, by the increasing complexities in conducting clinical research, and by a lack of dedicated child-health research funding. Given these obstacles, pediatric translational research can be enhanced by developing strategic and efficient biobanks that will provide scientists with quality tissue specimens to render accurate and reproducible research results. Indeed, tissue sampling and biobanking within pediatric academic settings has potential to impact child health by promoting bidirectional interaction between clinicians and scientists, helping to maximize research productivity, and providing a competitive edge for attracting and maintaining high-quality personnel. The authors of this review outline key issues and practical solutions to optimize pediatric tissue sampling and biobanking for translational research, activities that will ultimately reduce the burden of childhood disease.

  13. Light propagation in tissues: effect of finite size of tissue sample

    NASA Astrophysics Data System (ADS)

    Melnik, Ivan S.; Dets, Sergiy M.; Rusina, Tatyana V.

    1995-12-01

    Laser beam propagation inside tissues with different lateral dimensions has been considered. Scattering and anisotropic properties of tissue critically determine spatial fluence distribution and predict sizes of tissue specimens when deviations of this distribution can be neglected. Along the axis of incident beam the fluence rate weakly depends on sample size whereas its relative increase (more than 20%) towards the lateral boundaries. The finite sizes were considered to be substantial only for samples with sizes comparable with the diameter of the laser beam. Interstitial irradiance patterns simulated by Monte Carlo method were compared with direct measurements in human brain specimens.

  14. Ultra-trace analysis of platinum in human tissue samples.

    PubMed

    Rudolph, Elisabeth; Hann, Stephan; Stingeder, Gerhard; Reiter, Christian

    2005-08-01

    Background levels of platinum were determined in human autopsy tissues taken from five individuals. The investigated specimens were lung, liver and kidney. Sample preparation involved microwave digestion followed by an open vessel treatment. Inductively-coupled plasma sector field mass spectrometry (ICP-SFMS) was applied in combination with an ultrasonic nebulization/membrane desolvation system for sample introduction. Isotope dilution analysis was employed for accurate quantification of platinum. Excellent procedural detection limits (3 s validation) of 20, 20 and 34 pg g(-1) dry weight were obtained for lung, liver and kidney tissue, respectively. Due to the lack of appropriate biological reference material, road dust (BCR-723) was used for method validation. Platinum levels ranging between 0.03 and 1.42 ng g(-1) were determined in the investigated samples. The platinum concentrations observed in human lung tissue may reflect the increasing atmospheric background levels of platinum originating from car catalysts. The presence of platinum in kidney and liver tissue samples clearly indicates the bioavailability of the element.

  15. Building additional complexity to in vitro-derived intestinal tissues.

    PubMed

    Brugmann, Samantha A; Wells, James M

    2013-01-01

    Gastrointestinal (GI) disorders affect up to 25% of the US population. Common intestinal disorders include malabsorption, irritable bowel syndrome and fecal incontinence. Some GI disorders such as Hirschsprung's disease have a genetic basis and are associated with an absence or paucity of enteric nerves. Current treatment plans for GI disorders range from changes in diet to bowel resection, and there are very few drugs available that target the primary deficiencies in intestinal function such as controlled peristalsis. While animal models can recapitulate the broad range of intestinal pathologies of the GI tract, they are intrinsically complicated and of low throughput. Several in vitro systems have been established, and these range from epithelial enteroids to more complex organoids, which contain most intestinal cell types. One of the more complex organoid systems was derived from adult mouse intestines and contains functional enteric nerves and smooth muscle capable of peristalsis. Establishing an equivalent human intestinal system is challenging due to limited access and variable quality of human intestinal tissues. However, owing to recent advances, it is possible to differentiate human induced and embryonic pluripotent stem cells, collectively called pluripotent stem cells, into human intestinal organoids (HIOs) in vitro. Although HIOs contain a significant degree of epithelial and mesenchymal complexity, they lack enteric nerves and thus are unable to model the peristaltic movements of the gut. The goal of this review is to discuss approaches to generate complex in vitro systems that can be used to more comprehensively model common intestinal pathologies. New and more biologically complete human models of the intestine would allow for unprecedented studies of the cellular and molecular basis of normal and pathological gut function. Furthermore, fully functional HIOs could serve as a platform for preclinical drug studies to model absorption and efficacy.

  16. 52 additional reference population samples for the 55 AISNP panel.

    PubMed

    Pakstis, Andrew J; Haigh, Eva; Cherni, Lotfi; ElGaaied, Amel Ben Ammar; Barton, Alison; Evsanaa, Baigalmaa; Togtokh, Ariunaa; Brissenden, Jane; Roscoe, Janet; Bulbul, Ozlem; Filoglu, Gonul; Gurkan, Cemal; Meiklejohn, Kelly A; Robertson, James M; Li, Cai-Xia; Wei, Yi-Liang; Li, Hui; Soundararajan, Usha; Rajeevan, Haseena; Kidd, Judith R; Kidd, Kenneth K

    2015-11-01

    Ancestry inference for a person using a panel of SNPs depends on the variation of frequencies of those SNPs around the world and the amount of reference data available for calculation/comparison. The Kidd Lab panel of 55 AISNPs has been incorporated in commercial kits by both Life Technologies and Illumina for massively parallel sequencing. Therefore, a larger set of reference populations will be useful for researchers using those kits. We have added reference population allele frequencies for 52 population samples to the 73 previously entered so that there are now allele frequencies publicly available in ALFRED and FROG-kb for a total of 125 population samples.

  17. Proteomic analysis of tissue samples in translational breast cancer research.

    PubMed

    Gromov, Pavel; Moreira, José M A; Gromova, Irina

    2014-06-01

    In the last decade, many proteomic technologies have been applied, with varying success, to the study of tissue samples of breast carcinoma for protein expression profiling in order to discover protein biomarkers/signatures suitable for: characterization and subtyping of tumors; early diagnosis, and both prognosis and prediction of outcome of chemotherapy. The purpose of this review is to critically appraise what has been achieved to date using proteomic technologies and to bring forward novel strategies - based on the analysis of clinically relevant samples - that promise to accelerate the translation of basic discoveries into the daily breast cancer clinical practice. In particular, we address major issues in experimental design by reviewing the strengths and weaknesses of current proteomic strategies in the context of the analysis of human breast tissue specimens.

  18. Sex identification of polar bears from blood and tissue samples

    USGS Publications Warehouse

    Amstrup, Steven C.; Garner, G.W.; Cronin, M.A.; Patton, J.C.

    1993-01-01

    Polar bears (Ursus maritimus) can be adversely affected by hunting and other human perturbations because of low population densities and low reproduction rates. The sustainable take of adult females may be as low as 1.5% of the population. Females and accompanying young are most vulnerable to hunting, and hunters have not consistently reported the sex composition of the harvest, therefore a method to confirm the sexes of polar bears harvested in Alaska is needed. Evidence of the sex of harvested animals is often not available, but blood or other tissue samples often are. We extracted DNA from tissue and blood samples, and amplified segments of zinc finger (ZFX and ZFY) genes from both X and Y chromosomes with the polymerase chain reaction. Digestion of amplified portions of the X chromosome with the restriction enzyme HaeIII resulted in subdivision of the original amplified segment into four smaller fragments. Digestion with HaeIII did not subdivide the original segment amplified from the Y chromosome. The differing fragment sizes produced patterns in gel electrophoresis that distinguished samples from male and female bears 100% of the time. This technique is applicable to the investigation of many wildlife management and research questions.

  19. The visual assessment of broth cultures for tissue bank samples.

    PubMed

    Varettas, Kerry

    2017-01-05

    The bioburden screening process of allograft musculoskeletal tissue samples received at the South Eastern Area Laboratory Services includes the routine use of solid agar and cooked meat (CM) broth media. CM has been routinely sub-cultured onto solid agar plates after aerobic incubation at 35 °C. This study will evaluate whether a visual assessment of CM can replace sub-culture by an in vitro inoculation and a prospective study. Eight challenge organisms were serially diluted and inoculated into CM. The average inoculum of 0.5-5.5 CFU produced visible turbidity of CM after 24-h incubation for 7 of the challenge organisms with one organism producing turbidity after 48-h incubation. The prospective study evaluated 222 CM of which 213 were visually clear and no-growth on sub-culture and 9 turbid CM which were culture positive. Broth cultures are an integral part of the bioburden screening process of allograft musculoskeletal tissue and swab samples and visual assessment of CM can replace sub-culture.

  20. Analysis of dissected tissues with digital holographic microscopy: quantification of inflammation mediated tissue alteration, influence of sample preparation, and reliability of numerical autofocusing

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Lenz, Philipp; Bettenworth, Dominik; Krausewitz, Philipp; Domagk, Dirk; Ketelhut, Steffi

    2015-03-01

    Quantitative phase imaging with digital holographic microscopy (DHM) allows label-free imaging of tissue sections and quantification of the spatial refractive index distribution, which is of interest for applications in digital pathology. We show that DHM allows quantitative imaging of different layers in unstained tissue samples by detection of refractive index changes. In addition, we evaluate the automated refocussing feature of DHM for application on dissected tissues and could achieve highly reproducible holographic autofocusing for unstained and moderately stained samples. Finally, it is demonstrated that in human ulcerative colitis patients the average tissue refractive index is reduced significantly in all parts of the inflamed colonic wall in comparison to patients in remission.

  1. Effects of formalin fixation on tissue optical properties of in-vitro brain samples

    NASA Astrophysics Data System (ADS)

    Anand, Suresh; Cicchi, Riccardo; Martelli, Fabrizio; Giordano, Flavio; Buccoliero, Anna Maria; Guerrini, Renzo; Pavone, Francesco S.

    2015-03-01

    Application of light spectroscopy based techniques for the detection of cancers have emerged as a promising approach for tumor diagnostics. In-vivo or freshly excised samples are normally used for point spectroscopic studies. However, ethical issues related to in-vivo studies, rapid decay of surgically excised tissues and sample availability puts a limitation on in-vivo and in-vitro studies. There has been a few studies reported on the application of formalin fixed samples with good discrimination capability. Usually formalin fixation is performed to prevent degradation of tissues after surgical resection. Fixing tissues in formalin prevents cell death by forming cross-linkages with proteins. Previous investigations have revealed that washing tissues fixed in formalin using phosphate buffered saline is known to reduce the effects of formalin during spectroscopic measurements. But this could not be the case with reflectance measurements. Hemoglobin is a principal absorbing medium in biological tissues in the visible range. Formalin fixation causes hemoglobin to seep out from red blood cells. Also, there could be alterations in the refractive index of tissues when fixed in formalin. In this study, we propose to investigate the changes in tissue optical properties between freshly excised and formalin fixed brain tissues. The results indicate a complete change in the spectral profile in the visible range where hemoglobin has its maximum absorption peaks. The characteristic bands of oxy-hemoglobin at 540, 580 nm and deoxy-hemoglobin at 555 nm disappear in the case of samples fixed in formalin. In addition, an increased spectral intensity was observed for the wavelengths greater than 650 nm where scattering phenomena are presumed to dominate.

  2. Immunohistochemical detection of Aspergillus species in pediatric tissue samples.

    PubMed

    Choi, John K; Mauger, Joanne; McGowan, Karin L

    2004-01-01

    Definitive diagnosis of invasive aspergillosis often requires tissue samples for histologic evidence of fungal infection and culture confirmation of Aspergillus species. However, the culture frequently fails to isolate Aspergillus species. Alternative approaches to confirm Aspergillus infection use polymerase chain reaction, in situ hybridization, and immunohistochemical analysis on paraffin-embedded sections. These approaches are well characterized in animals and adult patients but not pediatric patients. We studied the immunoreactivity of a commercially available monoclonal antibody, Mab-WF-AF-1 (DAKO, Carpinteria, CA), on paraffin-embedded sections from 16 pediatric cases with invasive aspergillosis, of which 12 were proven by culture. Optimal immunoreactivity required microwave antigen retrieval using high pH; 5 other antigen retrieval approaches were unsuccessful. With optimization, the monoclonal antibody was strongly immunoreactive in all cases with staining of the Aspergillus cell wall, septa, and cytoplasm. Background was minimal with no cross-reactivity to Candida albicans. These findings demonstrate the usefulness of the Mab-WF-AF-1 antibody in pediatric tissues suspected of invasive aspergillosis.

  3. Measurement of phthalates in small samples of mammalian tissue

    SciTech Connect

    Acott, P.D.; Murphy, M.G.; Ogborn, M.R.; Crocker, J.F.S.

    1987-03-01

    Di-(2-ethylhexyl)-phthalate (DEHP) is a phthalic acid ester that is used as a plasticizer in polyvinyl chloride products, many of which have widespread medical application. DEHP has been shown to be leached from products used for storage and delivery of blood transfusions during procedures such as plasmaphoresis, hemodialysis and open heart surgery. Results of studies in this laboratory have suggested that there is an association between the absorption and deposition of DEHP (and/or related chemicals) in the kidney and the acquired renal cystic disease (ACD) frequently seen in patients who have undergone prolonged dialysis treatment. In order to determine the relationship between the two, it has been necessary to establish a method for extracting and accurately quantitating minute amounts of these chemicals in small tissue samples. The authors have now established such a method using kidneys from normal rats and from a rat model for ACD.

  4. Discriminating healthy from tumor and necrosis tissue in rat brain tissue samples by Raman spectral imaging.

    PubMed

    Amharref, Nadia; Beljebbar, Abdelilah; Dukic, Sylvain; Venteo, Lydie; Schneider, Laurence; Pluot, Michel; Manfait, Michel

    2007-10-01

    The purpose of this study was to investigate molecular changes associated with glioma tissues by Raman microspectroscopy in order to develop its use in clinical practice. Spectroscopic markers obtained from C6 glioma tissues were compared to conventional histological and histochemical techniques. Cholesterol and phospholipid contents were highest in corpus callosum and decreased gradually towards the cortex surface as well as in the tumor. Two different necrotic areas have been identified: a fully necrotic zone characterized by the presence of plasma proteins and a peri-necrotic area with a high lipid content. This result was confirmed by Nile Red staining. Additionally, one structure was detected in the periphery of the tumor. Invisible with histopathological hematoxylin and eosin staining, it was revealed by immunohistochemical Ki-67 and MT1-MMP staining used to visualize the proliferative and invasive activities of glioma, respectively. Hierarchical cluster analysis on the only cluster averaged spectra showed a clear distinction between normal, tumoral, necrotic and edematous tissues. Raman microspectroscopy can discriminate between healthy and tumoral brain tissue and yield spectroscopic markers associated with the proliferative and invasive properties of glioblastoma. Development of in vivo Raman spectroscopy could thus accurately define tumor margins, identify tumor remnants, and help in the development of novel therapies for glioblastoma.

  5. Cancer Detection in Human Tissue Samples Using a Fiber-Tip pH Probe.

    PubMed

    Schartner, Erik P; Henderson, Matthew R; Purdey, Malcolm; Dhatrak, Deepak; Monro, Tanya M; Gill, P Grantley; Callen, David F

    2016-12-01

    Intraoperative detection of tumorous tissue is an important unresolved issue for cancer surgery. Difficulty in differentiating between tissue types commonly results in the requirement for additional surgeries to excise unremoved cancer tissue or alternatively in the removal of excess amounts of healthy tissue. Although pathologic methods exist to determine tissue type during surgery, these methods can compromise postoperative pathology, have a lag of minutes to hours before the surgeon receives the results of the tissue analysis, and are restricted to excised tissue. In this work, we report the development of an optical fiber probe that could potentially find use as an aid for margin detection during surgery. A fluorophore-doped polymer coating is deposited on the tip of an optical fiber, which can then be used to record the pH by monitoring the emission spectra from this dye. By measuring the tissue pH and comparing with the values from regular tissue, the tissue type can be determined quickly and accurately. The use of a novel lift-and-measure technique allows for these measurements to be performed without influence from the inherent autofluorescence that commonly affects fluorescence-based measurements on biological samples. The probe developed here shows strong potential for use during surgery, as the probe design can be readily adapted to a low-cost portable configuration, which could find use in the operating theater. Use of this probe in surgery either on excised or in vivo tissue has the potential to improve success rates for complete removal of cancers. Cancer Res; 76(23); 6795-801. ©2016 AACR.

  6. Optimization of Evans blue quantitation in limited rat tissue samples

    NASA Astrophysics Data System (ADS)

    Wang, Hwai-Lee; Lai, Ted Weita

    2014-10-01

    Evans blue dye (EBD) is an inert tracer that measures plasma volume in human subjects and vascular permeability in animal models. Quantitation of EBD can be difficult when dye concentration in the sample is limited, such as when extravasated dye is measured in the blood-brain barrier (BBB) intact brain. The procedure described here used a very small volume (30 µl) per sample replicate, which enabled high-throughput measurements of the EBD concentration based on a standard 96-well plate reader. First, ethanol ensured a consistent optic path length in each well and substantially enhanced the sensitivity of EBD fluorescence spectroscopy. Second, trichloroacetic acid (TCA) removed false-positive EBD measurements as a result of biological solutes and partially extracted EBD into the supernatant. Moreover, a 1:2 volume ratio of 50% TCA ([TCA final] = 33.3%) optimally extracted EBD from the rat plasma protein-EBD complex in vitro and in vivo, and 1:2 and 1:3 weight-volume ratios of 50% TCA optimally extracted extravasated EBD from the rat brain and liver, respectively, in vivo. This procedure is particularly useful in the detection of EBD extravasation into the BBB-intact brain, but it can also be applied to detect dye extravasation into tissues where vascular permeability is less limiting.

  7. Phase-contrast Hounsfield units of fixated and non-fixated soft-tissue samples

    SciTech Connect

    Willner, Marian; Fior, Gabriel; Marschner, Mathias; Birnbacher, Lorenz; Schock, Jonathan; Braun, Christian; Fingerle, Alexander A.; Noël, Peter B.; Rummeny, Ernst J.; Pfeiffer, Franz; Herzen, Julia; Rozhkova, Elena A.

    2015-08-31

    X-ray phase-contrast imaging is a novel technology that achieves high soft-tissue contrast. Although its clinical impact is still under investigation, the technique may potentially improve clinical diagnostics. In conventional attenuation-based X-ray computed tomography, radiological diagnostics are quantified by Hounsfield units. Corresponding Hounsfield units for phase-contrast imaging have been recently introduced, enabling a setup-independent comparison and standardized interpretation of imaging results. Thus far, the experimental values of few tissue types have been reported; these values have been determined from fixated tissue samples. This study presents phase-contrast Hounsfield units for various types of non-fixated human soft tissues. A large variety of tissue specimens ranging from adipose, muscle and connective tissues to liver, kidney and pancreas tissues were imaged by a grating interferometer with a rotating-anode X-ray tube and a photon-counting detector. In addition, we investigated the effects of formalin fixation on the quantitative phase-contrast imaging results.

  8. Phase-contrast Hounsfield units of fixated and non-fixated soft-tissue samples

    DOE PAGES

    Willner, Marian; Fior, Gabriel; Marschner, Mathias; ...

    2015-08-31

    X-ray phase-contrast imaging is a novel technology that achieves high soft-tissue contrast. Although its clinical impact is still under investigation, the technique may potentially improve clinical diagnostics. In conventional attenuation-based X-ray computed tomography, radiological diagnostics are quantified by Hounsfield units. Corresponding Hounsfield units for phase-contrast imaging have been recently introduced, enabling a setup-independent comparison and standardized interpretation of imaging results. Thus far, the experimental values of few tissue types have been reported; these values have been determined from fixated tissue samples. This study presents phase-contrast Hounsfield units for various types of non-fixated human soft tissues. A large variety of tissuemore » specimens ranging from adipose, muscle and connective tissues to liver, kidney and pancreas tissues were imaged by a grating interferometer with a rotating-anode X-ray tube and a photon-counting detector. In addition, we investigated the effects of formalin fixation on the quantitative phase-contrast imaging results.« less

  9. Preservation and rapid purification of DNA from decomposing human tissue samples.

    PubMed

    Sorensen, Amy; Rahman, Elizabeth; Canela, Cassandra; Gangitano, David; Hughes-Stamm, Sheree

    2016-11-01

    One of the key features to be considered in a mass disaster is victim identification. However, the recovery and identification of human remains are sometimes complicated by harsh environmental conditions, limited facilities, loss of electricity and lack of refrigeration. If human remains cannot be collected, stored, or identified immediately, bodies decompose and DNA degrades making genotyping more difficult and ultimately decreasing DNA profiling success. In order to prevent further DNA damage and degradation after collection, tissue preservatives may be used. The goal of this study was to evaluate three customized (modified TENT, DESS, LST) and two commercial DNA preservatives (RNAlater and DNAgard(®)) on fresh and decomposed human skin and muscle samples stored in hot (35°C) and humid (60-70% relative humidity) conditions for up to three months. Skin and muscle samples were harvested from the thigh of three human cadavers placed outdoors for up to two weeks. In addition, the possibility of purifying DNA directly from the preservative solutions ("free DNA") was investigated in order to eliminate lengthy tissue digestion processes and increase throughput. The efficiency of each preservative was evaluated based on the quantity of DNA recovered from both the "free DNA" in solution and the tissue sample itself in conjunction with the quality and completeness of downstream STR profiles. As expected, DNA quantity and STR success decreased with time of decomposition. However, a marked decrease in DNA quantity and STR quality was observed in all samples after the bodies entered the bloat stage (approximately six days of decomposition in this study). Similar amounts of DNA were retrieved from skin and muscle samples over time, but slightly more complete STR profiles were obtained from muscle tissue. Although higher amounts of DNA were recovered from tissue samples than from the surrounding preservative, the average number of reportable alleles from the "free DNA" was

  10. Screening of Viral Pathogens from Pediatric Ileal Tissue Samples after Vaccination

    DOE PAGES

    Hewitson, Laura; Thissen, James B.; Gardner, Shea N.; ...

    2014-01-01

    In 2010, researchers reported that the two US-licensed rotavirus vaccines contained DNA or DNA fragments from porcine circovirus (PCV). Although PCV, a common virus among pigs, is not thought to cause illness in humans, these findings raised several safety concerns. In this study, we sought to determine whether viruses, including PCV, could be detected in ileal tissue samples of children vaccinated with one of the two rotavirus vaccines. A broad spectrum, novel DNA detection technology, the Lawrence Livermore Microbial Detection Array (LLMDA), was utilized, and confirmation of viral pathogens using the polymerase chain reaction (PCR) was conducted. The LLMDAmore » technology was recently used to identify PCV from one rotavirus vaccine. Ileal tissue samples were analyzed from 21 subjects, aged 15–62 months. PCV was not detected in any ileal tissue samples by the LLMDA or PCR. LLMDA identified a human rotavirus A from one of the vaccinated subjects, which is likely due to a recent infection from a wild type rotavirus. LLMDA also identified human parechovirus, a common gastroenteritis viral infection, from two subjects. Additionally, LLMDA detected common gastrointestinal bacterial organisms from the Enterobacteriaceae , Bacteroidaceae , and Streptococcaceae families from several subjects. This study provides a survey of viral and bacterial pathogens from pediatric ileal samples, and may shed light on future studies to identify pathogen associations with pediatric vaccinations.« less

  11. Screening of Viral Pathogens from Pediatric Ileal Tissue Samples after Vaccination

    PubMed Central

    Thissen, James B.; Gardner, Shea N.; McLoughlin, Kevin S.; Glausser, Margaret K.; Jaing, Crystal J.

    2014-01-01

    In 2010, researchers reported that the two US-licensed rotavirus vaccines contained DNA or DNA fragments from porcine circovirus (PCV). Although PCV, a common virus among pigs, is not thought to cause illness in humans, these findings raised several safety concerns. In this study, we sought to determine whether viruses, including PCV, could be detected in ileal tissue samples of children vaccinated with one of the two rotavirus vaccines. A broad spectrum, novel DNA detection technology, the Lawrence Livermore Microbial Detection Array (LLMDA), was utilized, and confirmation of viral pathogens using the polymerase chain reaction (PCR) was conducted. The LLMDA technology was recently used to identify PCV from one rotavirus vaccine. Ileal tissue samples were analyzed from 21 subjects, aged 15–62 months. PCV was not detected in any ileal tissue samples by the LLMDA or PCR. LLMDA identified a human rotavirus A from one of the vaccinated subjects, which is likely due to a recent infection from a wild type rotavirus. LLMDA also identified human parechovirus, a common gastroenteritis viral infection, from two subjects. Additionally, LLMDA detected common gastrointestinal bacterial organisms from the Enterobacteriaceae, Bacteroidaceae, and Streptococcaceae families from several subjects. This study provides a survey of viral and bacterial pathogens from pediatric ileal samples, and may shed light on future studies to identify pathogen associations with pediatric vaccinations. PMID:24778651

  12. MicroRNA Stability in FFPE Tissue Samples: Dependence on GC Content

    PubMed Central

    Kakimoto, Yu; Tanaka, Masayuki; Kamiguchi, Hiroshi; Ochiai, Eriko; Osawa, Motoki

    2016-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs responsible for fine-tuning of gene expression at post-transcriptional level. The alterations in miRNA expression levels profoundly affect human health and often lead to the development of severe diseases. Currently, high throughput analyses, such as microarray and deep sequencing, are performed in order to identify miRNA biomarkers, using archival patient tissue samples. MiRNAs are more robust than longer RNAs, and resistant to extreme temperatures, pH, and formalin-fixed paraffin-embedding (FFPE) process. Here, we have compared the stability of miRNAs in FFPE cardiac tissues using next-generation sequencing. The mode read length in FFPE samples was 11 nucleotides (nt), while that in the matched frozen samples was 22 nt. Although the read counts were increased 1.7-fold in FFPE samples, compared with those in the frozen samples, the average miRNA mapping rate decreased from 32.0% to 9.4%. These results indicate that, in addition to the fragmentation of longer RNAs, miRNAs are to some extent degraded in FFPE tissues as well. The expression profiles of total miRNAs in two groups were highly correlated (0.88 tissues instead of miR-1, which was predominant before fixation. Subsequent quantitative PCR (qPCR) analyses revealed that miRNAs with GC content of less than 40% are more degraded than GC-rich miRNAs (p<0.0001). We showed that deep sequencing data obtained using FFPE samples cannot be directly compared with that of fresh frozen samples. The combination of miRNA deep sequencing and other quantitative analyses, such as qPCR, may improve the utility of archival FFPE tissue samples. PMID:27649415

  13. Quantitative laser-induced breakdown spectroscopy analysis of calcified tissue samples

    NASA Astrophysics Data System (ADS)

    Samek, O.; Beddows, D. C. S.; Telle, H. H.; Kaiser, J.; Liška, M.; Cáceres, J. O.; Gonzáles Ureña, A.

    2001-06-01

    We report on the application of laser-induced breakdown spectroscopy (LIBS) to the analysis of important minerals and the accumulation of potentially toxic elements in calcified tissue, to trace e.g. the influence of environmental exposure, and other medical or biological factors. This theme was exemplified for quantitative detection and mapping of Al, Pb and Sr in representative samples, including teeth (first teeth of infants, second teeth of children and teeth of adults) and bones (tibia and femur). In addition to identifying and quantifying major and trace elements in the tissues, one- and two-dimensional profiles and maps were generated. Such maps (a) provide time/concentration relations, (b) allow to follow mineralisation of the hydroxyapatite matrix and the migration of the elements within it and (c) enable to identify disease states, such as caries in teeth. In order to obtain quantitative calibration, reference samples in the form of pressed pellets with calcified tissue-equivalent material (majority compound of pellets is CaCO 3) were used whose physical properties closely resembled hydroxyapatite. Compounds of Al, Sr and Pb were added to the pellets, containing atomic concentrations in the range 100-10 000 ppm relative to the Ca content of the matrix. Analytical results based on this calibration against artificial samples for the trace elements under investigation agree with literature values, and with our atomic absorption spectroscopy (AAS) cross-validation measurements.

  14. Studying Genes in Tissue Samples From Younger and Adolescent Patients With Soft Tissue Sarcomas

    ClinicalTrials.gov

    2016-05-13

    Childhood Alveolar Soft-part Sarcoma; Childhood Angiosarcoma; Childhood Desmoplastic Small Round Cell Tumor; Childhood Epithelioid Sarcoma; Childhood Fibrosarcoma; Childhood Leiomyosarcoma; Childhood Liposarcoma; Childhood Malignant Mesenchymoma; Childhood Neurofibrosarcoma; Childhood Synovial Sarcoma; Chordoma; Desmoid Tumor; Metastatic Childhood Soft Tissue Sarcoma; Nonmetastatic Childhood Soft Tissue Sarcoma; Recurrent Childhood Soft Tissue Sarcoma

  15. 40 CFR 80.8 - Sampling methods for gasoline, diesel fuel, fuel additives, and renewable fuels.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... fuel, fuel additives, and renewable fuels. 80.8 Section 80.8 Protection of Environment ENVIRONMENTAL... Provisions § 80.8 Sampling methods for gasoline, diesel fuel, fuel additives, and renewable fuels. The..., blendstocks, fuel additives and renewable fuels for purposes of determining compliance with the...

  16. Molecular classification of cancer with the 92-gene assay in cytology and limited tissue samples

    PubMed Central

    Brachtel, Elena F.; Operaña, Theresa N.; Sullivan, Peggy S.; Kerr, Sarah E.; Cherkis, Karen A.; Schroeder, Brock E.; Dry, Sarah M.; Schnabel, Catherine A.

    2016-01-01

    Background Detailed molecular evaluation of cytology and limited tissue samples is increasingly becoming the standard for cancer care. Reproducible and accurate diagnostic approaches with reduced demands on cellularity are an ongoing unmet need. This study evaluated the performance of a 92-gene assay for molecular diagnosis of tumor type/subtype in cytology and limited tissue samples. Methods Clinical validation of accuracy for the 92-gene assay in limited tissue samples such as cytology cell blocks, core biopsies and small excisions was conducted in a blinded multi-institutional study (N = 109, 48% metastatic, 53% grade II and III). Analytical success rate and diagnostic utility were evaluated in a consecutive series of 644 cytology cases submitted for clinical testing. Results The 92-gene assay demonstrated 91% sensitivity (95% CI [0.84, 0.95]) for tumor classification, with high accuracy maintained irrespective of specimen type (100%, 92%, and 86% in FNA/cytology cell blocks, core biopsies, and small excisions, respectively; p = 0.26). The assay performed equally well for metastatic versus primary tumors (90% vs 93%, p = 0.73), and across histologic grades (100%, 90%, 89%, in grades I, II, and III, respectively; p = 0.75). In the clinical case series, a molecular diagnosis was reported in 87% of the 644 samples, identifying 23 different tumor types and allowing for additional mutational analysis in selected cases. Conclusions These findings demonstrate high accuracy and analytical success rate of the 92-gene assay, supporting its utility in the molecular diagnosis of cancer for specimens with limited tissue. PMID:27034010

  17. Stability of ethyl glucuronide in urine, post-mortem tissue and blood samples.

    PubMed

    Schloegl, Haiko; Dresen, Sebastian; Spaczynski, Karin; Stoertzel, Mylène; Wurst, Friedrich Martin; Weinmann, Wolfgang

    2006-03-01

    The stability of ethyl glucuronide (EtG) under conditions of degradation was examined in urine samples of nine volunteers and in post-mortem tissue (liver, skeletal muscle) and blood taken from seven corpses at autopsies. Analysis was performed via LC-MS/MS. EtG concentrations in urine samples ranged from 2.5 to 296.5 mg/l. When stored at 4 degrees C in airtight test tubes, EtG concentrations remained relatively constant; when stored at room temperature (RT) for 5 weeks in ventilated vials, variations of EtG concentrations ranged from a 30% decrease to an 80% increase, with an average of 37.5% increase. Liver and skeletal muscle tissue of three corpses with positive blood alcohol concentrations (BAC; ranging from 0.106 to 0.183 g%) were stored for 4 weeks and analysed periodically. EtG concentrations decreased 27.7% on average in 4 weeks storage at RT but EtG was still detectable in all samples with initial EtG concentrations higher than 1 mug/g. Blood and liver samples of four corpses with negative BACs were stored at RT after addition of 0.1 g% ethanol, and no new formation of EtG was observed.

  18. 49 CFR 199.111 - Retention of samples and additional testing.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... SAFETY DRUG AND ALCOHOL TESTING Drug Testing § 199.111 Retention of samples and additional testing. (a...) Since some analytes may deteriorate during storage, detected levels of the drug below the...

  19. 49 CFR 199.111 - Retention of samples and additional testing.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... SAFETY DRUG AND ALCOHOL TESTING Drug Testing § 199.111 Retention of samples and additional testing. (a...) Since some analytes may deteriorate during storage, detected levels of the drug below the...

  20. How does tissue regeneration influence the mechanical behavior of additively manufactured porous biomaterials?

    PubMed

    Hedayati, R; Janbaz, S; Sadighi, M; Mohammadi-Aghdam, M; Zadpoor, A A

    2017-01-01

    Although the initial mechanical properties of additively manufactured porous biomaterials are intensively studied during the last few years, almost no information is available regarding the evolution of the mechanical properties of implant-bone complex as the tissue regeneration progresses. In this paper, we studied the effects of tissue regeneration on the static and fatigue behavior of selective laser melted porous titanium structures with three different porosities (i.e. 77, 81, and 85%). The porous structures were filled with four different polymeric materials with mechanical properties in the range of those observed for de novo bone (0.7GPatissue regeneration into their pores. The static mechanical properties and fatigue behavior (S-N curves) of as-manufactured and filled porous structures were then determined. The static mechanical properties and fatigue life (including endurance limit) of the porous structures were found to increase by factors 2-7, even when they were filled with polymeric materials with relatively low mechanical properties. The relative increase in the mechanical properties was much higher for the porous structures with lower porosities. Moreover, the increase in the fatigue life was more notable as compared to the increase in the static mechanical properties. Such large values of increase in the mechanical properties with the progress of bone tissue regeneration have implications in terms of mechanical stimulus for bone tissue regeneration.

  1. Additives

    NASA Technical Reports Server (NTRS)

    Smalheer, C. V.

    1973-01-01

    The chemistry of lubricant additives is discussed to show what the additives are chemically and what functions they perform in the lubrication of various kinds of equipment. Current theories regarding the mode of action of lubricant additives are presented. The additive groups discussed include the following: (1) detergents and dispersants, (2) corrosion inhibitors, (3) antioxidants, (4) viscosity index improvers, (5) pour point depressants, and (6) antifouling agents.

  2. Effects of various dietary lipid additives on lamb performance, carcass characteristics, adipose tissue fatty acid composition, and wool characteristics.

    PubMed

    Meale, S J; Chaves, A V; He, M L; Guan, L L; McAllister, T A

    2015-06-01

    Tasco (Ascophyllum nodosum; TA) was compared to canola (CO), flax (FO), and safflower oils (SO) for effects on performance, carcass characteristics, and fatty acid profiles of adipose tissue in skirt muscle (SM), subcutaneous and perirenal adipose tissues, and wool production and quality characteristics of Canadian Arcott lambs. Fifty-six lambs were randomly assigned to dietary treatments (n = 14 per treatment). Diets consisted of a pelleted, barley-based finishing diet containing either TA, CO, FO, or SO (2% of dietary DM). Feed deliveries and orts were recorded daily. Lambs were weighed weekly and slaughtered once they reached ≥ 45 kg BW. Carcass characteristics, rumen pH, and liver weights were determined at slaughter. Wool yield was determined on mid-side patches of 100 cm2 shorn at d 0 and on the day before slaughter (d 105 or 140). Dye-bands were used to determine wool growth, micrometer and staple length. Adipose tissues and SM samples were taken at slaughter and analyzed for FA profiles. No effects were observed on intake, growth, or carcass characteristics. A greater (P = 0.02) staple strength of lambs fed CO was the only effect observed in wool. Flax oil increased total n-3 and decreased the n-6/n-3 ratio in tissue FA profiles (P < 0.001) in comparison to other diets. Tasco increased (P ≤ 0.001) SFA/PUFA in all tissues, whereas concentrations of CLA c-9, t-11 were greatest with SO in all tissues (P ≤ 0.02), compared to other diets. These results suggest Tasco supplementation did not improve the n-3/n-6 or SFA/PUFA ratios of lamb adipose tissues compared to other dietary lipid additives.

  3. Final LDRD report : development of sample preparation methods for ChIPMA-based imaging mass spectrometry of tissue samples.

    SciTech Connect

    Maharrey, Sean P.; Highley, Aaron M.; Behrens, Richard, Jr.; Wiese-Smith, Deneille

    2007-12-01

    The objective of this short-term LDRD project was to acquire the tools needed to use our chemical imaging precision mass analyzer (ChIPMA) instrument to analyze tissue samples. This effort was an outgrowth of discussions with oncologists on the need to find the cellular origin of signals in mass spectra of serum samples, which provide biomarkers for ovarian cancer. The ultimate goal would be to collect chemical images of biopsy samples allowing the chemical images of diseased and nondiseased sections of a sample to be compared. The equipment needed to prepare tissue samples have been acquired and built. This equipment includes an cyro-ultramicrotome for preparing thin sections of samples and a coating unit. The coating unit uses an electrospray system to deposit small droplets of a UV-photo absorbing compound on the surface of the tissue samples. Both units are operational. The tissue sample must be coated with the organic compound to enable matrix assisted laser desorption/ionization (MALDI) and matrix enhanced secondary ion mass spectrometry (ME-SIMS) measurements with the ChIPMA instrument Initial plans to test the sample preparation using human tissue samples required development of administrative procedures beyond the scope of this LDRD. Hence, it was decided to make two types of measurements: (1) Testing the spatial resolution of ME-SIMS by preparing a substrate coated with a mixture of an organic matrix and a bio standard and etching a defined pattern in the coating using a liquid metal ion beam, and (2) preparing and imaging C. elegans worms. Difficulties arose in sectioning the C. elegans for analysis and funds and time to overcome these difficulties were not available in this project. The facilities are now available for preparing biological samples for analysis with the ChIPMA instrument. Some further investment of time and resources in sample preparation should make this a useful tool for chemical imaging applications.

  4. Rapid microarray-based genotyping of Chlamydia spp. strains from clinical tissue samples.

    PubMed

    Sachse, Konrad; Ruettger, Anke

    2015-01-01

    Pathogenic Chlamydia (C.) psittaci and C. trachomatis strains can be genotyped based on variations in the ompA genomic locus. In the present chapter, we describe rapid genotyping assays for both chlamydial agents using the ArrayStrip™ (AS) microarray platform. The test is targeting multiple discriminatory sites in the variable domains of the ompA gene by using 35 (C. psittaci) and 61 (C. trachomatis) oligonucleotide probes representing genotype-specific polymorphisms. In addition to discrimination among the established genotypes, this approach allows identification of atypical strains that were not accessible to typing using previously established techniques, such as PCR-RFLP or serotyping. The present DNA microarray assay can be conducted directly on clinical tissue samples and is suitable for tracing epidemiological chains and exploring the dissemination of particular genotypes. The procedure is easy to handle and economically affordable, and it allows genotyping of up to 32 clinical samples per day, thus lending itself for routine diagnosis as well.

  5. Micro-organisms isolated from cadaveric samples of allograft musculoskeletal tissue.

    PubMed

    Varettas, Kerry

    2013-12-01

    Allograft musculoskeletal tissue is commonly used in orthopaedic surgical procedures. Cadaveric donors of musculoskeletal tissue supply multiple allografts such as tendons, ligaments and bone. The microbiology laboratory of the South Eastern Area Laboratory Services (SEALS, Australia) has cultured cadaveric allograft musculoskeletal tissue samples for bacterial and fungal isolates since 2006. This study will retrospectively review the micro-organisms isolated over a 6-year period, 2006-2011. Swab and tissue samples were received for bioburden testing and were inoculated onto agar and/or broth culture media. Growth was obtained from 25.1 % of cadaveric allograft musculoskeletal tissue samples received. The predominant organisms isolated were coagulase-negative staphylococci and coliforms, with the heaviest bioburden recovered from the hemipelvis. The rate of bacterial and fungal isolates from cadaveric allograft musculoskeletal tissue samples is higher than that from living donors. The type of organism isolated may influence the suitability of the allograft for transplant.

  6. An additive manufacturing-based PCL-alginate-chondrocyte bioprinted scaffold for cartilage tissue engineering.

    PubMed

    Kundu, Joydip; Shim, Jin-Hyung; Jang, Jinah; Kim, Sung-Won; Cho, Dong-Woo

    2015-11-01

    Regenerative medicine is targeted to improve, restore or replace damaged tissues or organs using a combination of cells, materials and growth factors. Both tissue engineering and developmental biology currently deal with the process of tissue self-assembly and extracellular matrix (ECM) deposition. In this investigation, additive manufacturing (AM) with a multihead deposition system (MHDS) was used to fabricate three-dimensional (3D) cell-printed scaffolds using layer-by-layer (LBL) deposition of polycaprolactone (PCL) and chondrocyte cell-encapsulated alginate hydrogel. Appropriate cell dispensing conditions and optimum alginate concentrations for maintaining cell viability were determined. In vitro cell-based biochemical assays were performed to determine glycosaminoglycans (GAGs), DNA and total collagen contents from different PCL-alginate gel constructs. PCL-alginate gels containing transforming growth factor-β (TGFβ) showed higher ECM formation. The 3D cell-printed scaffolds of PCL-alginate gel were implanted in the dorsal subcutaneous spaces of female nude mice. Histochemical [Alcian blue and haematoxylin and eosin (H&E) staining] and immunohistochemical (type II collagen) analyses of the retrieved implants after 4 weeks revealed enhanced cartilage tissue and type II collagen fibril formation in the PCL-alginate gel (+TGFβ) hybrid scaffold. In conclusion, we present an innovative cell-printed scaffold for cartilage regeneration fabricated by an advanced bioprinting technology.

  7. Elemental distribution and sample integrity comparison of freeze-dried and frozen-hydrated biological tissue samples with nuclear microprobe

    NASA Astrophysics Data System (ADS)

    Vavpetič, P.; Vogel-Mikuš, K.; Jeromel, L.; Ogrinc Potočnik, N.; Pongrac, P.; Drobne, D.; Pipan Tkalec, Ž.; Novak, S.; Kos, M.; Koren, Š.; Regvar, M.; Pelicon, P.

    2015-04-01

    The analysis of biological samples in frozen-hydrated state with micro-PIXE technique at Jožef Stefan Institute (JSI) nuclear microprobe has matured to a point that enables us to measure and examine frozen tissue samples routinely as a standard research method. Cryotome-cut slice of frozen-hydrated biological sample is mounted between two thin foils and positioned on the sample holder. The temperature of the cold stage in the measuring chamber is kept below 130 K throughout the insertion of the samples and the proton beam exposure. Matrix composition of frozen-hydrated tissue is consisted mostly of ice. Sample deterioration during proton beam exposure is monitored during the experiment, as both Elastic Backscattering Spectrometry (EBS) and Scanning Transmission Ion Microscopy (STIM) in on-off axis geometry are recorded together with the events in two PIXE detectors and backscattered ions from the chopper in a single list-mode file. The aim of this experiment was to determine differences and similarities between two kinds of biological sample preparation techniques for micro-PIXE analysis, namely freeze-drying and frozen-hydrated sample preparation in order to evaluate the improvements in the elemental localisation of the latter technique if any. In the presented work, a standard micro-PIXE configuration for tissue mapping at JSI was used with five detection systems operating in parallel, with proton beam cross section of 1.0 × 1.0 μm2 and a beam current of 100 pA. The comparison of the resulting elemental distributions measured at the biological tissue prepared in the frozen-hydrated and in the freeze-dried state revealed differences in elemental distribution of particular elements at the cellular level due to the morphology alteration in particular tissue compartments induced either by water removal in the lyophilisation process or by unsatisfactory preparation of samples for cutting and mounting during the shock-freezing phase of sample preparation.

  8. [PCR-based diagnosis of mucormycosis in tissue samples].

    PubMed

    Bialek, R; Zelck, U E

    2013-11-01

    Mucormycosis is characterized by a rapid, often fatal progression. Early diagnosis of invasive mucormycosis is the key for timely therapeutic intervention and improved survival. Contrary to the more prevalent aspergillosis, effective antifungal therapy of mucormycosis is mainly limited to amphotericin B. Given the importance to guide the timely initiation of amphotericin B and possible surgical intervention, rapid and specific identification of fungal hyphae is essential. Conventional histopathology depends on abundance and morphology of the fungi as well as on the skills of the personnel, and usually shows an accuracy of 80 %. PCR assays targeting fungal ribosomal genes to identify Mucorales at least at genus level increase sensitivity, allow a rapid identification as well as detection of double mold infections. Thus, PCR assays are beneficial to complement existing approaches. They are recommended to rapidly specify tissue diagnosis and accurate identification of fungi. This will help to guide effective therapy and thereby, survival will increase. Retrospective analyses of mucormycosis by PCR help to evaluate therapeutic interventions and will optimize treatment options.

  9. Monitoring the marine environment using marine mammal tissue samples

    SciTech Connect

    Jones, P.D.; Hannah, D.J.; Day, P.J.

    1995-12-31

    Marine environments, both inshore and open ocean, receive numerous inputs of anthropogenic chemicals. Cetaceans provide a valuable resource for monitoring the low level contamination of marine environments with persistent organic contaminants. Comparative studies using inshore and offshore southern ocean cetaceans have revealed significant differences in the types of contamination in these two environments. The polychlorinated biphenyls (PCBs) deposited in the southern oceans are characterized by an abundance of lower chlorinated congeners. Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F) are not present at significant concentrations in cetaceans from the open southern ocean. In contrast significant concentrations of PCDD/F congeners are detected in the blubber of the inshore living Hector`s dolphin. This species lives close to the shore and has a very small home range (approximately 30 km) for a cetacean. Analysis of tissue PCDD/F and PCB profiles from different populations and their food sources will be presented. The data are being used to determine if there are local variations in the contamination of the New Zealand inshore marine environment.

  10. Cytokine assays: an assessment of the preparation and treatment of blood and tissue samples.

    PubMed

    Keustermans, Genoveva C E; Hoeks, Sanne B E; Meerding, Jenny M; Prakken, Berent J; de Jager, Wilco

    2013-05-15

    Cytokines are key components of the innate and adaptive immune system. As pivotal players in the progression or regression of a pathological process, these molecules provide a window through which diseases can be monitored and can thus act as biomarkers. In order to measure cytokine levels, a plethora of protocols can be applied. These methods include bioassays, protein microarrays, high-performance liquid chromatography (HPLC), sandwich enzyme-linked immunosorbent assay (ELISA), Meso Scale Discovery (MSD) electrochemiluminescence and bead based multiplex immunoassays (MIA). Due to the interaction and activity of cytokines, multiplex immunoassays are at the forefront of cytokine analysis by allowing multiple cytokines to be measured in parallel. However, even with optimized protocols, sample standardization needs to occur before these proteins can optimally act as biomarkers. This review describes various factors influencing the levels of cytokines measured in plasma, serum, dried blood spots and tissue biopsies, focusing on sample collection and handling, long term storage and the repetitive use of samples. By analyzing how each of these factors influences protein levels, it is concluded that samples should be stored at low temperatures in order to maintain cytokine stability. In addition, within a study, sample manipulations should be kept the same, with measurement protocols being chosen for their compatibility with the research in question. By having a clear understanding of what factors influence cytokine levels and how to overcome these technical issues, minimally confounded data can be obtained and cytokines can achieve optimal biomarker activity.

  11. A novel method for measuring aromatase activity in tissue samples by determining estradiol concentrations.

    PubMed

    Tinwell, H; Rascle, J B; Colombel, S; Al Khansa, I; Freyberger, A; Bars, R

    2011-07-01

    Increasing scrutiny of endocrine disrupters has led to changes to European pesticide and biocide legislation and to the introduction of the Endocrine Disrupter Screening Program by the US EPA. One element of endocrine disrupter identification is to determine its effects on aromatase, but most available assays are limited as they depend on tritiated water production to indicate enzyme activity. Whilst acceptable for determining aromatase effects using a cell-free approach, this method is unreliable for cell or tissue-based investigations as other cytochrome P-450 isoenzyme activities can similarly produce tritiated water and consequently confound interpretation of the aromatase data. To address this lack of specificity an assay directly measuring the final estrogen product by incubating rat tissue protein with testosterone and measuring the resultant estradiol concentration was developed. Using this approach we demonstrated marked increases in enzyme activity in pregnant rat ovary samples and dose-related inhibitions when incubating non-pregnant rat ovary samples with known aromatase inhibitors. Hepatic aromatase activity was investigated using our method and by tritiated water production with microsomes from rats dosed with the antiandrogen 1,1-dichloro-2,2-bis(4 chlorophenyl)ethane. Additional cytochrome P-450s were also measured. Treatment-related increased tritiated water production and general hepatic enzyme activity were recorded but estradiol was not increased, indicating that the increased tritiated water was due to general enzyme activity and not aromatase activity. A simple and specific method has been developed that can detect aromatase inhibition and induction, which when applied to tissue samples, provides a means of generating relevant animal data concerning chemical effects on the aromatase enzyme.

  12. Grinding and polishing instead of sectioning for the tissue samples with a graft: Implications for light and electron microscopy.

    PubMed

    Mukhamadiyarov, Rinat A; Sevostyanova, Victoria V; Shishkova, Daria K; Nokhrin, Andrey V; Sidorova, Olga D; Kutikhin, Anton G

    2016-06-01

    A broad use of the graft replacement requires a detailed investigation of the host-graft interaction, including both histological examination and electron microscopy. A high quality sectioning of the host tissue with a graft seems to be complicated; in addition, it is difficult to examine the same tissue area by both of the mentioned microscopy techniques. To solve these problems, we developed a new technique of epoxy resin embedding with the further grinding, polishing, and staining. Graft-containing tissues prepared by grinding and polishing preserved their structure; however, sectioning frequently required the explantation of the graft and led to tissue disintegration. Moreover, stained samples prepared by grinding and polishing may then be assessed by both light microscopy and backscattered scanning electron microscopy. Therefore, grinding and polishing outperform sectioning when applied to the tissues with a graft.

  13. Methods for using 3-D ultrasound speckle tracking in biaxial mechanical testing of biological tissue samples.

    PubMed

    Yap, Choon Hwai; Park, Dae Woo; Dutta, Debaditya; Simon, Marc; Kim, Kang

    2015-04-01

    Being multilayered and anisotropic, biological tissues such as cardiac and arterial walls are structurally complex, making the full assessment and understanding of their mechanical behavior challenging. Current standard mechanical testing uses surface markers to track tissue deformations and does not provide deformation data below the surface. In the study described here, we found that combining mechanical testing with 3-D ultrasound speckle tracking could overcome this limitation. Rat myocardium was tested with a biaxial tester and was concurrently scanned with high-frequency ultrasound in three dimensions. The strain energy function was computed from stresses and strains using an iterative non-linear curve-fitting algorithm. Because the strain energy function consists of terms for the base matrix and for embedded fibers, spatially varying fiber orientation was also computed by curve fitting. Using finite-element simulations, we first validated the accuracy of the non-linear curve-fitting algorithm. Next, we compared experimentally measured rat myocardium strain energy function values with those in the literature and found a matching order of magnitude. Finally, we retained samples after the experiments for fiber orientation quantification using histology and found that the results satisfactorily matched those computed in the experiments. We conclude that 3-D ultrasound speckle tracking can be a useful addition to traditional mechanical testing of biological tissues and may provide the benefit of enabling fiber orientation computation.

  14. Tissue targeted metabonomics: metabolic profiling by microdialysis sampling and microcoil NMR.

    PubMed

    Price, Kristin E; Vandaveer, Shannon S; Lunte, Craig E; Larive, Cynthia K

    2005-08-10

    The concentration of low molecular weight compounds in tissues can yield valuable information about the metabolic state of an organism. Studies of changes in the metabolic state or metabonomics can reflect disease pathways, drug action, or toxicity. This research aims to develop a new approach, tissue targeted metabonomics. Microdialysis sampling and microcoil NMR analysis are employed to compare basal and ischemic metabolic states of various tissues (blood, brain, and heart) of Sprague-Dawley rats. Microdialysis sampling is localized, making the metabolic profile tissue specific. Coupling to NMR analysis is highly advantageous, because a complete metabolic profile is obtained in a single spectrum. However, small sample volumes and low analyte concentrations make analysis of microdialysis samples challenging. Microcoil NMR uses low sample volumes and has improved mass sensitivity, relative to standard 5 mm probes. The coupling of these techniques is a potentially powerful tool for metabonomics analysis.

  15. 31. VIEW FROM SOUTHWEST TO CORNER WHERE SAMPLING/CRUSHING ADDITIONS ABUT ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    31. VIEW FROM SOUTHWEST TO CORNER WHERE SAMPLING/CRUSHING ADDITIONS ABUT CRUSHED OXIDIZED ORE BIN. INTACT BARREN SOLUTION TANK VISIBLE IN FRONT OF CRUSHED ORE BIN. - Bald Mountain Gold Mill, Nevada Gulch at head of False Bottom Creek, Lead, Lawrence County, SD

  16. Analysis of the amount of tissue sample necessary for mitotic count and Ki-67 index in gastrointestinal stromal tumor sampling.

    PubMed

    Kobara, Hideki; Mori, Hirohito; Rafiq, Kazi; Fujihara, Shintaro; Nishiyama, Noriko; Chiyo, Taiga; Matsunaga, Tae; Ayaki, Maki; Yachida, Tatsuo; Kato, Kiyohito; Kamada, Hideki; Fujita, Koji; Morishita, Asahiro; Oryu, Makoto; Tsutsui, Kunihiko; Iwama, Hisakazu; Kushida, Yoshio; Haba, Reiji; Masaki, Tsutomu

    2015-01-01

    There are no established opinions concerning whether the amount of tissue affects the accuracy of histological analyses in gastrointestinal stromal tumors (GISTs). The aim of the present study was to investigate the appropriate amount of tissue sample needed for mitotic count based on the risk classification of GISTs and the Ki-67 index using the following three methods: endoscopic ultrasound-guided fine-needle aspiration (FNA), a novel sampling method called tunneling bloc biopsy (TBB), and biopsy forceps followed by TBB (Bf). Forty-three samples (12 FNA, 17 TBB and 14 Bf) diagnosed as GISTs by immunohistological analysis were utilized. The major and minor axes and overlay area of one piece of specimen (OPS) from the three sampling methods were measured using digital imaging software and were analyzed comparatively regarding the acquisition of histological data. The mean major and minor axes (mm) and overlay areas (mm2) were in the order of TBB > Bf > FNA. The evaluable rates by mitotic count and Ki-67 were, respectively, 75% (9/12) and 83.3% (10/12) for FNA samples, 100% (17/17) and 100% (17/17) for TBB samples, and 100% (14/14) and 100% (14/14) for Bf samples (P>0.05). Three FNA samples were judged unevaluable due to too small specimens in overall diagnosis including mitotic count and Ki-67, calculating the cut-off value for the overlay area of OPS as 0.17 mm2. Comparing the concordance rates between the pre- and post-operative samples, TBB samples was significantly better than FNA (P<0.05). Conclusively, while the amounts of tissues obtained by TBB and Bf are unnecessary for the histological assessment of mitotic count and Ki-67 index, developments of the FNA method are needed to minimize sample error. Considering the technical aspects, as well as the size of the specimens, could help to guide therapeutic planning and improve diagnostic yield for GI subepithelial tumors.

  17. Studies of levels of biogenic amines in meat samples in relation to the content of additives.

    PubMed

    Jastrzębska, Aneta; Kowalska, Sylwia; Szłyk, Edward

    2016-01-01

    The impact of meat additives on the concentration of biogenic amines and the quality of meat was studied. Fresh white and red meat samples were fortified with the following food additives: citric and lactic acids, disodium diphosphate, sodium nitrite, sodium metabisulphite, potassium sorbate, sodium chloride, ascorbic acid, α-tocopherol, propyl 3,4,5-trihydroxybenzoate (propyl gallate) and butylated hydroxyanisole. The content of spermine, spermidine, putrescine, cadaverine, histamine, tyramine, tryptamine and 2-phenylethylamine was determined by capillary isotachophoretic methods in meat samples (fresh and fortified) during four days of storage at 4°C. The results were applied to estimate the impact of the tested additives on the formation of biogenic amines in white and red meat. For all tested meats, sodium nitrite, sodium chloride and disodium diphosphate showed the best inhibition. However, cadaverine and putrescine were characterised by the biggest changes in concentration during the storage time of all the additives. Based on the presented data for the content of biogenic amines in meat samples analysed as a function of storage time and additives, we suggest that cadaverine and putrescine have a significant impact on meat quality.

  18. Radiometric solvent-partitioning assay for screening cocaine hydrolases and measuring cocaine levels in milligram tissue samples.

    PubMed

    Brimijoin, Stephen; Shen, Maryann L; Sun, Hong

    2002-10-15

    To permit rapid screening and characterization of novel cocaine hydrolases, as well as accurate measurement of cocaine levels in small samples of tissue, a radiometric assay was developed. The assay is based on selective, organic solvent partition of [3H]benzene-labeled cocaine or of [3H]benzoic acid liberated during enzymatic hydrolysis. With dilute samples the assay can be conducted entirely in scintillation vials and quantitated by addition of appropriate aqueous buffer and toluene-based fluor, making phase separation unnecessary. In this way, several hundred samples can be assayed in an afternoon, nanogram quantities of enzyme can be characterized without prior purification, and cocaine concentrations can be accurately measured in milligram samples of tissue after administration of [3H]cocaine in vivo.

  19. 76 FR 24862 - Proposed Information Collection; Comment Request; Protocol for Access to Tissue Specimen Samples...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-03

    ... unusual mortality events through two projects, the Marine Mammal Health and Stranding Response Program... for Access to Tissue Specimen Samples From the National Marine Mammal Tissue Bank AGENCY: National... Patricia.Lawson@noaa.gov . SUPPLEMENTARY INFORMATION: I. Abstract In 1989, the National Marine...

  20. An efficient, reliable and inexpensive device for the rapid homogenization of multiple tissue samples by centrifugation.

    PubMed

    Ilyin, S E; Plata-Salamán, C R

    2000-02-15

    Homogenization of tissue samples is a common first step in the majority of current protocols for RNA, DNA, and protein isolation. This report describes a simple device for centrifugation-mediated homogenization of tissue samples. The method presented is applicable to RNA, DNA, and protein isolation, and we show examples where high quality total cell RNA, DNA, and protein were obtained from brain and other tissue samples. The advantages of the approach presented include: (1) a significant reduction in time investment relative to hand-driven or individual motorized-driven pestle homogenization; (2) easy construction of the device from inexpensive parts available in any laboratory; (3) high replicability in the processing; and (4) the capacity for the parallel processing of multiple tissue samples, thus allowing higher efficiency, reliability, and standardization.

  1. How You Can Help Medical Research: Donating Your Blood, Tissue, and Other Samples

    MedlinePlus

    National Cancer Institute How You Can Help Medical Research U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Donating Your Blood, Tissue, and Other Samples You have the choice to ...

  2. Effects of Re-heating Tissue Samples to Core Body Temperature on High-Velocity Ballistic Projectile-tissue Interactions.

    PubMed

    Humphrey, Caitlin; Henneberg, Maciej; Wachsberger, Christian; Maiden, Nicholas; Kumaratilake, Jaliya

    2017-02-23

    Damage produced by high-speed projectiles on organic tissue will depend on the physical properties of the tissues. Conditioning organic tissue samples to human core body temperature (37°C) prior to conducting ballistic experiments enables their behavior to closely mimic that of living tissues. To minimize autolytic changes after death, the tissues are refrigerated soon after their removal from the body and re-heated to 37°C prior to testing. This research investigates whether heating 50-mm-cube samples of porcine liver, kidney, and heart to 37°C for varying durations (maximum 7 h) can affect the penetration response of a high-speed, steel sphere projectile. Longer conditioning times for heart and liver resulted in a slight loss of velocity/energy of the projectile, but the reverse effect occurred for the kidney. Possible reasons for these trends include autolytic changes causing softening (heart and liver) and dehydration causing an increase in density (kidney).

  3. Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

    PubMed Central

    Fang, Su-Chiung; Lien, Yi-Chen; Yang, Ting-Ting; Ko, Swee-Suak

    2016-01-01

    Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals(calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning. This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 °C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies. PMID:27911377

  4. Culture methods of allograft musculoskeletal tissue samples in Australian bacteriology laboratories.

    PubMed

    Varettas, Kerry

    2013-12-01

    Samples of allograft musculoskeletal tissue are cultured by bacteriology laboratories to determine the presence of bacteria and fungi. In Australia, this testing is performed by 6 TGA-licensed clinical bacteriology laboratories with samples received from 10 tissue banks. Culture methods of swab and tissue samples employ a combination of solid agar and/or broth media to enhance micro-organism growth and maximise recovery. All six Australian laboratories receive Amies transport swabs and, except for one laboratory, a corresponding biopsy sample for testing. Three of the 6 laboratories culture at least one allograft sample directly onto solid agar. Only one laboratory did not use a broth culture for any sample received. An international literature review found that a similar combination of musculoskeletal tissue samples were cultured onto solid agar and/or broth media. Although variations of allograft musculoskeletal tissue samples, culture media and methods are used in Australian and international bacteriology laboratories, validation studies and method evaluations have challenged and supported their use in recovering fungi and aerobic and anaerobic bacteria.

  5. Mitochondrial Respiration Chain Enzymatic Activities in the Human Brain: Methodological Implications for Tissue Sampling and Storage.

    PubMed

    Ronsoni, Marcelo Fernando; Remor, Aline Pertile; Lopes, Mark William; Hohl, Alexandre; Troncoso, Iris H Z; Leal, Rodrigo Bainy; Boos, Gustavo Luchi; Kondageski, Charles; Nunes, Jean Costa; Linhares, Marcelo Neves; Lin, Kátia; Latini, Alexandra Susana; Walz, Roger

    2016-04-01

    Mitochondrial respiratory chain complexes enzymatic (MRCCE) activities were successfully evaluated in frozen brain samples. Epilepsy surgery offers an ethical opportunity to study human brain tissue surgically removed to treat drug resistant epilepsies. Epilepsy surgeries are done with hemodynamic and laboratory parameters to maintain physiology, but there are no studies analyzing the association among these parameters and MRCCE activities in the human brain tissue. We determined the intra-operative parameters independently associated with MRCCE activities in middle temporal neocortex (Cx), amygdala (AMY) and head of hippocampus (HIP) samples of patients (n = 23) who underwent temporal lobectomy using multiple linear regressions. MRCCE activities in Cx, AMY and HIP are differentially associated to trans-operative mean arterial blood pressure, O2 saturation, hemoglobin, and anesthesia duration to time of tissue sampling. The time-course between the last seizure occurrence and tissue sampling as well as the sample storage to biochemical assessments were also associated with enzyme activities. Linear regression models including these variables explain 13-17 % of MRCCE activities and show a moderate to strong effect (r = 0.37-0.82). Intraoperative hemodynamic and laboratory parameters as well as the time from last seizure to tissue sampling and storage time are associated with MRCCE activities in human samples from the Cx, AMYG and HIP. Careful control of these parameters is required to minimize confounding biases in studies using human brain samples collected from elective neurosurgery.

  6. Detection and Genotyping of Human Papillomaviruses from Archival Formalin-Fixed Tissue Samples.

    PubMed

    Van Doorslaer, Koenraad; Chen, Zigui; McBride, Alison A

    2016-11-18

    Pathology departments routinely process and store formalin-fixed, paraffin-embedded (FFPE) tissue samples for clinical diagnosis. These collections often contain decades' worth of samples and represent a treasure trove of specimens that can be analyzed for retrospective epidemiological studies, diagnostics, and pathogen discovery. Accurate amplification and sequencing of DNA from these samples is critical for the usability of these FFPE samples. Here we present a collection of protocols that describe extraction of DNA from FFPE tissues, PCR amplification of human papillomavirus DNA, and subsequent genotyping of the infecting virus. © 2016 by John Wiley & Sons, Inc.

  7. Bimodal Spectroscopy of Formalin Fixed Samples to Discriminate Dysplastic and Tumor Brain Tissues

    NASA Astrophysics Data System (ADS)

    Anand, S.; Cicchi, R.; Giordano, F.; Buccoliero, A. M.; Guerrini, R.; Pavone, F. S.

    2014-12-01

    Biomedical spectroscopy has gained attention in the past few years for disease diagnosis. Fluorescence and Raman spectroscopies provide finger-print information related to biochemical and morphological alterations when tissues progress from the normal to a malignant stage. Usually, freshly excised tissue specimens are preferred for bio-spectroscopic studies. However, ethical issues, sample availability and distance between the surgery room and the laboratory provide an impelling restriction for in-vitro spectroscopic studies using freshly excised samples. After surgical resection tissues are fixed in 4% formalin for histological studies under a light microscope. The process of fixation prevents degradation of tissues. In this study, we probe the use of formalin fixed sample for differentiating normal and dysplastic brain tissues using fluorescence and Raman spectroscopies. It was found that fluorescence spectral profile changes in the wavelength range from 550-750 nm between dysplastic and tumor samples. Also, significant differences were found in the Raman spectral profiles of such samples. The results indicate a potential diagnostic application of spectroscopy in formalin fixed brain samples for differentiating dysplastic and tumor brain tissues.

  8. Qualification of serological infectious disease assays for the screening of samples from deceased tissue donors.

    PubMed

    Kitchen, A D; Newham, J A

    2011-05-01

    Whilst some of the assays used for serological screening of post-mortem blood samples from deceased tissue donors in some countries have been specifically validated by the manufacturer for this purpose, a significant number of those currently in use globally have not. Although specificity has previously been considered a problem in the screening of such samples, we believe that ensuring sensitivity is more important. The aim of this study was to validate a broader range of assays for the screening of post-mortem blood samples from deceased tissue donors. Six microplate immunoassays currently in use within National Health Service Blood and Transplant (NHSBT) for the screening of blood, tissue and stem cell donations were included. Representative samples from confirmed positive donors were titrated in screen negative post-mortem samples in parallel with normal pooled negative serum to determine if there was any inhibition with the post-mortem samples. There were no significant differences seen (P < 0.005) between the dilution curves obtained for the positive samples diluted in post-mortem samples and normal pooled sera. Although small numbers of samples were studied, it can be surmised that the post-mortem blood samples from deceased tissue donors, collected according to United Kingdom guidelines, are a suitable substrate for the assays evaluated. No diminution of reactivity was seen when dilution with sera from deceased donors was compared to dilution using pooled serum from live donors. In the absence of genuine low titre positive post-mortem samples, the use of samples spiked with various levels of target material provides a means of qualifying serological screening assays used by NHSBT for the screening of post-mortem blood samples from deceased tissue donors.

  9. Sources of Technical Variability in Quantitative LC-MS Proteomics: Human Brain Tissue Sample Analysis.

    SciTech Connect

    Piehowski, Paul D.; Petyuk, Vladislav A.; Orton, Daniel J.; Xie, Fang; Moore, Ronald J.; Ramirez Restrepo, Manuel; Engel, Anzhelika; Lieberman, Andrew P.; Albin, Roger L.; Camp, David G.; Smith, Richard D.; Myers, Amanda J.

    2013-05-03

    To design a robust quantitative proteomics study, an understanding of both the inherent heterogeneity of the biological samples being studied as well as the technical variability of the proteomics methods and platform is needed. Additionally, accurately identifying the technical steps associated with the largest variability would provide valuable information for the improvement and design of future processing pipelines. We present an experimental strategy that allows for a detailed examination of the variability of the quantitative LC-MS proteomics measurements. By replicating analyses at different stages of processing, various technical components can be estimated and their individual contribution to technical variability can be dissected. This design can be easily adapted to other quantitative proteomics pipelines. Herein, we applied this methodology to our label-free workflow for the processing of human brain tissue. For this application, the pipeline was divided into four critical components: Tissue dissection and homogenization (extraction), protein denaturation followed by trypsin digestion and SPE clean-up (digestion), short-term run-to-run instrumental response fluctuation (instrumental variance), and long-term drift of the quantitative response of the LC-MS/MS platform over the 2 week period of continuous analysis (instrumental stability). From this analysis, we found the following contributions to variability: extraction (72%) >> instrumental variance (16%) > instrumental stability (8.4%) > digestion (3.1%). Furthermore, the stability of the platform and its’ suitability for discovery proteomics studies is demonstrated.

  10. Investigation of reflectance sampling depth in biological tissues for various common illumination/collection configurations.

    PubMed

    Zonios, George

    2014-09-01

    Knowledge of light penetration characteristics is very important in almost all studies in biomedical optics. In this work, the reflectance sampling depth in biological tissues was investigated using Monte Carlo simulations for various common illumination/collection configurations. The analysis shows that the average sampling depth can be described by two simple empirical analytical expressions over the entire typical ranges of absorption and scattering properties relevant to in vivo biological tissue, regardless of the specific illumination/collection configuration details. These results are promising and helpful for the quick, efficient, and accurate design of reflectance studies for various biological tissue applications.

  11. An Unsupervised MVA Method to Compare Specific Regions in Human Breast Tumor Tissue Samples Using ToF-SIMS

    PubMed Central

    Bluestein, Blake M.; Morrish, Fionnuala; Graham, Daniel J.; Guenthoer, Jamie; Hockenbery, David; Porter, Peggy; Gamble, Lara J.

    2016-01-01

    Imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS) and principal component analysis (PCA) were used to investigate two sets of pre- and post-chemotherapy human breast tumor tissue sections to characterize lipids associated with tumor metabolic flexibility and response to treatment. The micron spatial resolution imaging capability of ToF-SIMS provides a powerful approach to attain spatially-resolved molecular and cellular data from cancerous tissues not available with conventional imaging techniques. Three ca. 1 mm2 areas per tissue section were analyzed by stitching together 200 μm × 200 μm raster area scans. A method to isolate and analyze specific tissue regions of interest by utilizing PCA of ToF-SIMS images is presented, which allowed separation of cellularized areas from stromal areas. These PCA-generated regions of interest were then used as masks to reconstruct representative spectra from specifically stromal or cellular regions. The advantage of this unsupervised selection method is a reduction in scatter in the spectral PCA results when compared to analyzing all tissue areas or analyzing areas highlighted by a pathologist. Utilizing this method, stromal and cellular regions of breast tissue biopsies taken pre- versus post-chemotherapy demonstrate chemical separation using negatively-charged ion species. In this sample set, the cellular regions were predominantly all cancer cells. Fatty acids (i.e. palmitic, oleic, and stearic), monoacylglycerols, diacylglycerols and vitamin E profiles were distinctively different between the pre- and post-therapy tissues. These results validate a new unsupervised method to isolate and interpret biochemically distinct regions in cancer tissues using imaging ToF-SIMS data. In addition, the method developed here can provide a framework to compare a variety of tissue samples using imaging ToF-SIMS, especially where there is section-to-section variability that makes it difficult to use a serial hematoxylin and

  12. Coupled Analysis of In Vitro and Histology Tissue Samples to Quantify Structure-Function Relationship

    PubMed Central

    Acar, Evrim; Plopper, George E.; Yener, Bülent

    2012-01-01

    The structure/function relationship is fundamental to our understanding of biological systems at all levels, and drives most, if not all, techniques for detecting, diagnosing, and treating disease. However, at the tissue level of biological complexity we encounter a gap in the structure/function relationship: having accumulated an extraordinary amount of detailed information about biological tissues at the cellular and subcellular level, we cannot assemble it in a way that explains the correspondingly complex biological functions these structures perform. To help close this information gap we define here several quantitative temperospatial features that link tissue structure to its corresponding biological function. Both histological images of human tissue samples and fluorescence images of three-dimensional cultures of human cells are used to compare the accuracy of in vitro culture models with their corresponding human tissues. To the best of our knowledge, there is no prior work on a quantitative comparison of histology and in vitro samples. Features are calculated from graph theoretical representations of tissue structures and the data are analyzed in the form of matrices and higher-order tensors using matrix and tensor factorization methods, with a goal of differentiating between cancerous and healthy states of brain, breast, and bone tissues. We also show that our techniques can differentiate between the structural organization of native tissues and their corresponding in vitro engineered cell culture models. PMID:22479315

  13. Terahertz spectroscopy and detection of brain tumor in rat fresh-tissue samples

    NASA Astrophysics Data System (ADS)

    Yamaguchi, S.; Fukushi, Y.; Kubota, O.; Itsuji, T.; Yamamoto, S.; Ouchi, T.

    2015-03-01

    Terahertz (THz) spectroscopy and imaging of biomedical samples is expected to be an important application of THz analysis techniques. Identification and localization of tumor tissue, imaging of biological samples, and analysis of DNA by THz spectroscopy have been reported. THz time-domain spectroscopy (TDS) is useful for obtaining the refractive index over a broad frequency range. However, THz-TDS spectra of fresh tissue samples are sensitive to procedures such as sample preparation, and a standardized measurement protocol is required. Therefore, in this work, we establish a protocol for measurements of THz spectra of fresh tissue and demonstrate reliable detection of rat brain tumor tissue. We use a reflection THz-TDS system to measure the refractive index spectra of the samples mounted on a quartz plate. The tissue samples were measured immediately after sectioning to avoid sample denaturalization during storage. Special care was taken in THz data processing to eliminate parasitic reflections and reduce noise. The error level in our refractive index measurements was as low as 0.02 in the frequency range 0.8-1.5 THz. With increasing frequency, the refractive index in the tumor and normal regions monotonically decreased, similarly to water, and it was 0.02 higher in the tumor regions. The spectral data suggest that the tumor regions have higher water content. Hematoxylin-eosin stained images showed that increased cell density was also responsible for the observed spectral features. A set of samples from 10 rats showed consistent results. Our results suggest that reliable tumor detection in fresh tissue without pretreatment is possible with THz spectroscopy measurements. THz spectroscopy has the potential to become a real-time in vivo diagnostic method.

  14. Sample Preparation for Mass Spectrometry Imaging of Plant Tissues: A Review

    PubMed Central

    Dong, Yonghui; Li, Bin; Malitsky, Sergey; Rogachev, Ilana; Aharoni, Asaph; Kaftan, Filip; Svatoš, Aleš; Franceschi, Pietro

    2016-01-01

    Mass spectrometry imaging (MSI) is a mass spectrometry based molecular ion imaging technique. It provides the means for ascertaining the spatial distribution of a large variety of analytes directly on tissue sample surfaces without any labeling or staining agents. These advantages make it an attractive molecular histology tool in medical, pharmaceutical, and biological research. Likewise, MSI has started gaining popularity in plant sciences; yet, information regarding sample preparation methods for plant tissues is still limited. Sample preparation is a crucial step that is directly associated with the quality and authenticity of the imaging results, it therefore demands in-depth studies based on the characteristics of plant samples. In this review, a sample preparation pipeline is discussed in detail and illustrated through selected practical examples. In particular, special concerns regarding sample preparation for plant imaging are critically evaluated. Finally, the applications of MSI techniques in plants are reviewed according to different classes of plant metabolites. PMID:26904042

  15. Comparison of EGFR mutation rates in lung adenocarcinoma tissue and pleural effusion samples.

    PubMed

    Guan, Y; Wang, Z J; Wang, L Q; Hua, D F; Liu, J

    2016-04-04

    The goal of the current study was to investigate the differences in epidermal growth factor receptor (EGFR) mutation rates in tumor tissue and pleural effusion specimens from patients with lung adenocarcinoma. PCR amplification and gene sequencing were used to detect EGFR mutations in exons 18, 19, 20, and 21 in tumor tissue and pleural effusion samples from 50 patients with advanced lung adenocarcinoma. The EGFR mutation rate was 34.0% in tissue samples from patients with advanced lung adenocarcinoma. There were 11 cases with exon 19 mutations and 6 cases with exon 21 mutations. The EGFR mutation rate was 30.0% in pleural effusion specimens, including 10 cases with exon 19 mutation and 5 cases with exon 21 mutations. Although the tissue samples had a slightly higher mutation rate compared to the pleural effusion samples, the difference was not statistically significant. These results indicate that the EGFR mutation rate detected in pleural effusion specimens from patients with advanced lung adenocarcinoma is similar to that detected in tumor tissue samples. Therefore, pleural effusion specimens can potentially be used for EGFR mutation detection in advanced lung adenocarcinoma.

  16. On the asymptotic improvement of supervised learning by utilizing additional unlabeled samples - Normal mixture density case

    NASA Technical Reports Server (NTRS)

    Shahshahani, Behzad M.; Landgrebe, David A.

    1992-01-01

    The effect of additional unlabeled samples in improving the supervised learning process is studied in this paper. Three learning processes. supervised, unsupervised, and combined supervised-unsupervised, are compared by studying the asymptotic behavior of the estimates obtained under each process. Upper and lower bounds on the asymptotic covariance matrices are derived. It is shown that under a normal mixture density assumption for the probability density function of the feature space, the combined supervised-unsupervised learning is always superior to the supervised learning in achieving better estimates. Experimental results are provided to verify the theoretical concepts.

  17. Multiplex short tandem repeat amplification of low template DNA samples with the addition of proofreading enzymes.

    PubMed

    Davis, Carey P; Chelland, Lynzee A; Pavlova, Victoria R; Illescas, María J; Brown, Kelly L; Cruz, Tracey Dawson

    2011-05-01

    With <100 pg of template DNA, routine short tandem repeat (STR) analysis often fails, resulting in no or partial profiles and increased stochastic effects. To overcome this, some have investigated preamplification methods that include the addition of proofreading enzymes to the PCR cocktail. This project sought to determine whether adding proofreading polymerases directly in the STR amplification mixture would improve the reaction when little template DNA is available. Platinum Taq High Fidelity and GeneAmp High Fidelity were tested in Profiler Plus™ STR reactions alone and in combination with AmpliTaq(®) Gold. All reactions included the additional step of a post-PCR purification step. With both pristine low template DNA and casework samples, the addition of these polymerases resulted in comparable or no improvement in the STR amplification signal. Further, stochastic effects and artifacts were observed equally across all enzyme conditions. Based on these studies, the addition of these proofreading enzymes to a multiplex STR amplification is not recommended for low template DNA work.

  18. Falcon: Visual analysis of large, irregularly sampled, and multivariate time series data in additive manufacturing

    DOE PAGES

    Steed, Chad A.; Halsey, William; Dehoff, Ryan; ...

    2017-02-16

    Flexible visual analysis of long, high-resolution, and irregularly sampled time series data from multiple sensor streams is a challenge in several domains. In the field of additive manufacturing, this capability is critical for realizing the full potential of large-scale 3D printers. Here, we propose a visual analytics approach that helps additive manufacturing researchers acquire a deep understanding of patterns in log and imagery data collected by 3D printers. Our specific goals include discovering patterns related to defects and system performance issues, optimizing build configurations to avoid defects, and increasing production efficiency. We introduce Falcon, a new visual analytics system thatmore » allows users to interactively explore large, time-oriented data sets from multiple linked perspectives. Falcon provides overviews, detailed views, and unique segmented time series visualizations, all with adjustable scale options. To illustrate the effectiveness of Falcon at providing thorough and efficient knowledge discovery, we present a practical case study involving experts in additive manufacturing and data from a large-scale 3D printer. The techniques described are applicable to the analysis of any quantitative time series, though the focus of this paper is on additive manufacturing.« less

  19. Pathology Tissue-quantitative Mass Spectrometry Analysis to Profile Histone Post-translational Modification Patterns in Patient Samples.

    PubMed

    Noberini, Roberta; Uggetti, Andrea; Pruneri, Giancarlo; Minucci, Saverio; Bonaldi, Tiziana

    2016-03-01

    Histone post-translational modifications (hPTMs) generate a complex combinatorial code that has been implicated with various pathologies, including cancer. Dissecting such a code in physiological and diseased states may be exploited for epigenetic biomarker discovery, but hPTM analysis in clinical samples has been hindered by technical limitations. Here, we developed a method (PAThology tissue analysis of Histones by Mass Spectrometry - PAT-H-MS) that allows to perform a comprehensive, unbiased and quantitative MS-analysis of hPTM patterns on formalin-fixed paraffin-embedded (FFPE) samples. In pairwise comparisons, histone extracted from formalin-fixed paraffin-embedded tissues showed patterns similar to fresh frozen samples for 24 differentially modified peptides from histone H3. In addition, when coupled with a histone-focused version of the super-SILAC approach, this method allows the accurate quantification of modification changes among breast cancer patient samples. As an initial application of the PAThology tissue analysis of Histones by Mass Spectrometry method, we analyzed breast cancer samples, revealing significant changes in histone H3 methylation patterns among Luminal A-like and Triple Negative disease subtypes. These results pave the way for retrospective epigenetic studies that combine the power of MS-based hPTM analysis with the extensive clinical information associated with formalin-fixed paraffin-embedded archives.

  20. Ice-cap. A high-throughput method for capturing plant tissue samples for genotype analysis.

    PubMed

    Krysan, Patrick

    2004-07-01

    High-throughput genotype screening is rapidly becoming a standard research tool in the post-genomic era. A major bottleneck currently exists, however, that limits the utility of this approach in the plant sciences. The rate-limiting step in current high-throughput pipelines is that tissue samples from living plants must be collected manually, one plant at a time. In this article I describe a novel method for harvesting tissue samples from living seedlings that eliminates this bottleneck. The method has been named Ice-Cap to reflect the fact that ice is used to capture the tissue samples. The planting of seeds, growth of seedlings, and harvesting of tissue are all performed in a 96-well format. I demonstrate the utility of this system by using tissue harvested by Ice-Cap to genotype a population of Arabidopsis seedlings that is segregating a previously characterized mutation. Because the harvesting of tissue is performed in a nondestructive manner, plants with the desired genotype can be transferred to soil and grown to maturity. I also show that Ice-Cap can be used to analyze genomic DNA from rice (Oryza sativa) seedlings. It is expected that this method will be applicable to high-throughput screening with many different plant species, making it a useful technology for performing marker assisted selection.

  1. Quantitative mapping of collagen fiber alignment in thick tissue samples using transmission polarized-light microscopy

    NASA Astrophysics Data System (ADS)

    Yakovlev, Dmitry D.; Shvachkina, Marina E.; Sherman, Maria M.; Spivak, Andrey V.; Pravdin, Alexander B.; Yakovlev, Dmitry A.

    2016-07-01

    Immersion optical clearing makes it possible to use transmission polarized-light microscopy for characterization of thick (200 to 2000 μm) layers of biological tissues. We discuss polarization properties of thick samples in the context of the problem of characterization of collagen fiber alignment in connective tissues such as sclera and dermis. Optical chirality caused by azimuthal variations of the macroscopic (effective) optic axis of the medium across the sample thickness should be considered in polarization mapping of thick samples of these tissues. We experimentally evaluate to what extent the optical chirality affects the measurement results in typical situations and show under what conditions it can be easily taken into account and does not hinder, but rather helps, in characterization of collagen fiber alignment.

  2. Effect of sample geometry on the apparent biaxial mechanical behaviour of planar connective tissues.

    PubMed

    Waldman, Stephen D; Lee, J Michael

    2005-12-01

    Mechanical testing methodologies developed for engineering materials may result in artifactual material properties if applied to soft planar connective tissues. The use of uniaxial tissue samples with high aspect ratios or biaxial samples with slender cruciform arms could lead to preferential loading of only the discrete subset of extracellular fibres that fully extend between the grips. To test this hypothesis, cruciform biaxial connective tissue samples that display distinctly different material properties (bovine pericardium, fish skin), as well as model textile laminates with predefined fibrous orientations, were repeatedly tested with decreasing sample arm lengths. With mechanical properties determined at the sample centre, results demonstrated that the materials appeared to become stiffer and less extensible with less slender sample geometries, suggesting that fibre recruitment increases with decreasing sample arm length. Alterations in the observed shear behaviour and rigid body rotation were also noted. The only truly reliable method to determine material properties is through in vivo testing, but this is not always convenient and is typically experimentally demanding. For the in vitro determination of the biaxial material properties, appropriate sample geometry should be employed in which all of the fibres contribute to the mechanical response.

  3. Statistical inference for the additive hazards model under outcome-dependent sampling.

    PubMed

    Yu, Jichang; Liu, Yanyan; Sandler, Dale P; Zhou, Haibo

    2015-09-01

    Cost-effective study design and proper inference procedures for data from such designs are always of particular interests to study investigators. In this article, we propose a biased sampling scheme, an outcome-dependent sampling (ODS) design for survival data with right censoring under the additive hazards model. We develop a weighted pseudo-score estimator for the regression parameters for the proposed design and derive the asymptotic properties of the proposed estimator. We also provide some suggestions for using the proposed method by evaluating the relative efficiency of the proposed method against simple random sampling design and derive the optimal allocation of the subsamples for the proposed design. Simulation studies show that the proposed ODS design is more powerful than other existing designs and the proposed estimator is more efficient than other estimators. We apply our method to analyze a cancer study conducted at NIEHS, the Cancer Incidence and Mortality of Uranium Miners Study, to study the risk of radon exposure to cancer.

  4. Statistical inference for the additive hazards model under outcome-dependent sampling

    PubMed Central

    Yu, Jichang; Liu, Yanyan; Sandler, Dale P.; Zhou, Haibo

    2015-01-01

    Cost-effective study design and proper inference procedures for data from such designs are always of particular interests to study investigators. In this article, we propose a biased sampling scheme, an outcome-dependent sampling (ODS) design for survival data with right censoring under the additive hazards model. We develop a weighted pseudo-score estimator for the regression parameters for the proposed design and derive the asymptotic properties of the proposed estimator. We also provide some suggestions for using the proposed method by evaluating the relative efficiency of the proposed method against simple random sampling design and derive the optimal allocation of the subsamples for the proposed design. Simulation studies show that the proposed ODS design is more powerful than other existing designs and the proposed estimator is more efficient than other estimators. We apply our method to analyze a cancer study conducted at NIEHS, the Cancer Incidence and Mortality of Uranium Miners Study, to study the risk of radon exposure to cancer. PMID:26379363

  5. Prospective Collection of Tissue Samples at Autopsy in Children with Diffuse Intrinsic Pontine Glioma

    PubMed Central

    Broniscer, Alberto; Baker, Justin N.; Baker, Suzanne J.; Chi, Susan N.; Geyer, J. Russell; Morris, E. Brannon; Gajjar, Amar

    2010-01-01

    BACKGROUND Brain tissue obtained at autopsy has been used in research for non-oncological disorders. However, this tool has never been systematically used in large investigational studies for cancer. We conducted a prospective, multicenter study to assess the feasibility of tissue collection at autopsy and its suitability for molecular analyses in children with diffuse intrinsic pontine glioma. METHODS Tumor tissue was collected at diagnosis, if clinically indicated, or at autopsy. Normal brain tissue was also collected at autopsy. The integrity of DNA and RNA was evaluated in all samples. Logistical data about autopsies were recorded. The feasibility of tissue collection at autopsy was assessed for patients treated at a single institution over a 43-month period. RESULTS Tumor samples were collected at diagnosis (n=3) or at autopsy (n=38) at 29 centers across the US; samples were obtained at diagnosis and autopsy in two cases. The median interval from death to autopsy was 7.7 hours. DNA and RNA with minimal or partial degradation, which were suitable for genome-wide analysis, were obtained from 100% and 63% of tumor samples, respectively. At the coordinating institution, approximately 40% of parents consented to autopsy and 40% declined. During the study period, 12 autopsies were obtained from patients who did not receive therapy at the coordinating center. CONCLUSIONS Multicenter, biological studies based on tissue obtained at autopsy are feasible in children with brain cancer. Our experience established a new paradigm for brain tissue collection which may increase the potential for research studies in patients with cancer. PMID:20589749

  6. Collecting and Storing Tissue, Blood, and Bone Marrow Samples From Patients With Rhabdomyosarcoma or Other Soft Tissue Sarcoma

    ClinicalTrials.gov

    2016-09-23

    Adult Rhabdomyosarcoma; Childhood Desmoplastic Small Round Cell Tumor; Chordoma; Desmoid Tumor; Metastatic Childhood Soft Tissue Sarcoma; Nonmetastatic Childhood Soft Tissue Sarcoma; Previously Treated Childhood Rhabdomyosarcoma; Previously Untreated Childhood Rhabdomyosarcoma; Recurrent Adult Soft Tissue Sarcoma; Recurrent Childhood Rhabdomyosarcoma; Recurrent Childhood Soft Tissue Sarcoma; Stage I Adult Soft Tissue Sarcoma; Stage II Adult Soft Tissue Sarcoma; Stage III Adult Soft Tissue Sarcoma; Stage IV Adult Soft Tissue Sarcoma

  7. Automated MALDI Matrix Coating System for Multiple Tissue Samples for Imaging Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Mounfield, William P.; Garrett, Timothy J.

    2012-03-01

    Uniform matrix deposition on tissue samples for matrix-assisted laser desorption/ionization (MALDI) is key for reproducible analyte ion signals. Current methods often result in nonhomogenous matrix deposition, and take time and effort to produce acceptable ion signals. Here we describe a fully-automated method for matrix deposition using an enclosed spray chamber and spray nozzle for matrix solution delivery. A commercial air-atomizing spray nozzle was modified and combined with solenoid controlled valves and a Programmable Logic Controller (PLC) to control and deliver the matrix solution. A spray chamber was employed to contain the nozzle, sample, and atomized matrix solution stream, and to prevent any interference from outside conditions as well as allow complete control of the sample environment. A gravity cup was filled with MALDI matrix solutions, including DHB in chloroform/methanol (50:50) at concentrations up to 60 mg/mL. Various samples (including rat brain tissue sections) were prepared using two deposition methods (spray chamber, inkjet). A linear ion trap equipped with an intermediate-pressure MALDI source was used for analyses. Optical microscopic examination showed a uniform coating of matrix crystals across the sample. Overall, the mass spectral images gathered from tissues coated using the spray chamber system were of better quality and more reproducible than from tissue specimens prepared by the inkjet deposition method.

  8. Automated MALDI matrix coating system for multiple tissue samples for imaging mass spectrometry.

    PubMed

    Mounfield, William P; Garrett, Timothy J

    2012-03-01

    Uniform matrix deposition on tissue samples for matrix-assisted laser desorption/ionization (MALDI) is key for reproducible analyte ion signals. Current methods often result in nonhomogenous matrix deposition, and take time and effort to produce acceptable ion signals. Here we describe a fully-automated method for matrix deposition using an enclosed spray chamber and spray nozzle for matrix solution delivery. A commercial air-atomizing spray nozzle was modified and combined with solenoid controlled valves and a Programmable Logic Controller (PLC) to control and deliver the matrix solution. A spray chamber was employed to contain the nozzle, sample, and atomized matrix solution stream, and to prevent any interference from outside conditions as well as allow complete control of the sample environment. A gravity cup was filled with MALDI matrix solutions, including DHB in chloroform/methanol (50:50) at concentrations up to 60 mg/mL. Various samples (including rat brain tissue sections) were prepared using two deposition methods (spray chamber, inkjet). A linear ion trap equipped with an intermediate-pressure MALDI source was used for analyses. Optical microscopic examination showed a uniform coating of matrix crystals across the sample. Overall, the mass spectral images gathered from tissues coated using the spray chamber system were of better quality and more reproducible than from tissue specimens prepared by the inkjet deposition method.

  9. Postmortem tissue samples: an alternative to urine and blood for drug analysis in racehorses.

    PubMed

    Uboh, C E; Rudy, J A; Railing, F A; Enright, J M; Shoemaker, J M; Kahler, M C; Shellenberger, J M; Kemecsei, Z; Das, D N

    1995-09-01

    Although urine is the sample of choice for drug tests in racehorses, it is rarely obtained following the sudden death of a racehorse on the track while racing. The purpose of this study was to demonstrate the significance of postmortem tissue samples as an alternative to urine and blood samples in equine drug analysis following the sudden death of a racehorse on the track while participating in a competitive race. Postmortem tissue samples were frozen (-80 degrees C) until analyzed. A 30-40-g portion of each organ was homogenized in a 0.1 M phosphate buffer (pH 7.4), deproteinized, hydrolyzed with beta-glucuronidase, extracted, and screened by thin-layer chromatography and immunoassay. Samples that initially tested positive for drug(s) were then extracted using high-flow, solid-phase extraction cartridges. The eluates were analyzed by gas chromatography-mass spectrometry. The presence of butorphanol in horses HB355 and CD387, pentobarbital in horse HO940, and ergotamine in horses HO940 and CD387 was detected and confirmed. Thus, in the absence of urine and blood samples following sudden death, postmortem tissue samples are equally useful for forensic toxicological investigations of racehorses.

  10. Assessing organic contaminants in fish: comparison of a nonlethal tissue sampling technique to mobile and stationary passive sampling devices.

    PubMed

    Heltsley, Rebecca M; Cope, W Gregory; Shea, Damian; Bringolf, Robert B; Kwak, Thomas J; Malindzak, Edward G

    2005-10-01

    As concerns mount over the human health risks associated with consumption of fish contaminated with persistent organic pollutants, there exists a need to better evaluate fish body burdens without lethally sampling many of the important commercial and sport species of interest. The aim of this study was to investigate two novel methods for estimating organic contaminants in fish that are a concern for both fish and human health. The removal of fish adipose fins, commonly done in mark-recapture studies with salmonid species, was evaluated as a nonlethal sampling technique to estimate concentrations of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) in flathead catfish (Pylodictis olivaris), relative to those found in muscle fillets of the same fish. We also assessed the efficacy of using poly(dimethylsiloxane) (PDMS) as a mobile passive sampling device (PSD) attached directly to wild flathead catfish for assessing location-specific exposure of the fish to waterborne contaminants. The results of this study have demonstrated for the first time that organic contaminant concentrations in adipose fin were highly correlated (R2 = 0.87) with muscle fillet concentrations, indicating that the adipose fin of certain fishes may be used to accurately estimate tissue concentrations without the need for lethal sampling. Moreover, mobile PSDs attached directly to fish and used here for the first time accurately estimated ultratrace concentrations of waterborne PCBs and OCPs without any apparent harm to the fish, indicating that there are no practical or physical barriers to the use of mobile passive samplers attached to aquatic organisms. Among the many practical implications of this research, two potential priority items include the analysis of organic contaminants in farm-raised and sport fish intended for human consumption, without the economic and population losses associated with lethally sampling fish to obtain tissues, and identifying specific areas

  11. Semiquantitative determination of polychlorinated biphenyls in tissue samples by thin layer chromatography

    USGS Publications Warehouse

    Mulhern, B.M.; Cromartie, E.; Reichel, W.L.; Belisle, A.A.

    1971-01-01

    A method is described for the analysis of polychlorinated biphenyl (PCB) compounds in tissue samples. Cleanup by hexane-aceto-nitrile partitioning and Florisil column chromatography are performed on samples before oxidative treatment to convert DDE to DCBP. PCB components are then determined semi-quantitatively by TLC. No prior separation of PCB from chlorinated pesticides is required. The lower limit of sensitivity is 0.2 ?g.

  12. A review on powder-based additive manufacturing for tissue engineering: selective laser sintering and inkjet 3D printing

    NASA Astrophysics Data System (ADS)

    Farid Seyed Shirazi, Seyed; Gharehkhani, Samira; Mehrali, Mehdi; Yarmand, Hooman; Metselaar, Hendrik Simon Cornelis; Adib Kadri, Nahrizul; Azuan Abu Osman, Noor

    2015-06-01

    Since most starting materials for tissue engineering are in powder form, using powder-based additive manufacturing methods is attractive and practical. The principal point of employing additive manufacturing (AM) systems is to fabricate parts with arbitrary geometrical complexity with relatively minimal tooling cost and time. Selective laser sintering (SLS) and inkjet 3D printing (3DP) are two powerful and versatile AM techniques which are applicable to powder-based material systems. Hence, the latest state of knowledge available on the use of AM powder-based techniques in tissue engineering and their effect on mechanical and biological properties of fabricated tissues and scaffolds must be updated. Determining the effective setup of parameters, developing improved biocompatible/bioactive materials, and improving the mechanical/biological properties of laser sintered and 3D printed tissues are the three main concerns which have been investigated in this article.

  13. A review on powder-based additive manufacturing for tissue engineering: selective laser sintering and inkjet 3D printing.

    PubMed

    Shirazi, Seyed Farid Seyed; Gharehkhani, Samira; Mehrali, Mehdi; Yarmand, Hooman; Metselaar, Hendrik Simon Cornelis; Adib Kadri, Nahrizul; Osman, Noor Azuan Abu

    2015-06-01

    Since most starting materials for tissue engineering are in powder form, using powder-based additive manufacturing methods is attractive and practical. The principal point of employing additive manufacturing (AM) systems is to fabricate parts with arbitrary geometrical complexity with relatively minimal tooling cost and time. Selective laser sintering (SLS) and inkjet 3D printing (3DP) are two powerful and versatile AM techniques which are applicable to powder-based material systems. Hence, the latest state of knowledge available on the use of AM powder-based techniques in tissue engineering and their effect on mechanical and biological properties of fabricated tissues and scaffolds must be updated. Determining the effective setup of parameters, developing improved biocompatible/bioactive materials, and improving the mechanical/biological properties of laser sintered and 3D printed tissues are the three main concerns which have been investigated in this article.

  14. A review on powder-based additive manufacturing for tissue engineering: selective laser sintering and inkjet 3D printing

    PubMed Central

    Shirazi, Seyed Farid Seyed; Gharehkhani, Samira; Mehrali, Mehdi; Yarmand, Hooman; Metselaar, Hendrik Simon Cornelis; Adib Kadri, Nahrizul; Osman, Noor Azuan Abu

    2015-01-01

    Since most starting materials for tissue engineering are in powder form, using powder-based additive manufacturing methods is attractive and practical. The principal point of employing additive manufacturing (AM) systems is to fabricate parts with arbitrary geometrical complexity with relatively minimal tooling cost and time. Selective laser sintering (SLS) and inkjet 3D printing (3DP) are two powerful and versatile AM techniques which are applicable to powder-based material systems. Hence, the latest state of knowledge available on the use of AM powder-based techniques in tissue engineering and their effect on mechanical and biological properties of fabricated tissues and scaffolds must be updated. Determining the effective setup of parameters, developing improved biocompatible/bioactive materials, and improving the mechanical/biological properties of laser sintered and 3D printed tissues are the three main concerns which have been investigated in this article. PMID:27877783

  15. Standardization of a sample preparation and analytical workflow for proteomics of archival endometrial cancer tissue.

    PubMed

    Alkhas, Addie; Hood, Brian L; Oliver, Kate; Teng, Pang-Ning; Oliver, Julie; Mitchell, David; Hamilton, Chad A; Maxwell, G Larry; Conrads, Thomas P

    2011-11-04

    The goal of the present study was to establish a standard operating procedure for mass spectrometry (MS)-based proteomic analysis of laser microdissected (LMD) formalin-fixed, paraffin-embedded (FFPE) uterine tissue. High resolution bioimage analysis of a large endometrial cancer tissue microarray immunostained for the breast cancer type 1 susceptibility protein enabled precise counting of cells to establish that there is an average of 600 cells/nL of endometrial cancer tissue. We sought to characterize the peptide recovery from various volumes of tissue gathered by LMD and processed/digested using the present methodology. We observed a nearly linear increase in peptide recovery amount with increasing tissue volume dissected. There was little discernible difference in the peptide recovery from stromal versus malignant epithelium, and there was no apparent difference in the day-to-day recovery. This methodology reproducibly results in 100 ng of digested peptides per nL of endometrial tissue, or ∼25 pg peptides/endometrial cancer cell. Results from liquid chromatography (LC)-MS/MS experiments to assess the impact of total peptide load on column on the total number of peptides and proteins identified from FFPE tissue digests prepared with the present methodology indicate a demonstrable increase in the total number of peptides identified up to 1000 ng, beyond which diminishing returns were observed. Furthermore, we observed no impact on the peptide identification rates from analyses of equivalent peptide amounts derived from lower volume LMD samples. These results show that this single-tube collection-to-injection proteomics (CTIP) workflow represents a straightforward, scalable, and highly reliable methodology for sample preparation to enable high throughput LMD-MS analysis of tissues derived from biopsy or surgery.

  16. Evaluation of two-dimensional electrophoresis and liquid chromatography – tandem mass spectrometry for tissue-specific protein profiling of laser-microdissected plant samples

    SciTech Connect

    Schad, Martina; Lipton, Mary S.; Giavalisco, Patrick; Smith, Richard D.; Kehr, Julia

    2005-07-14

    Laser microdissection (LM) allows the collection of homogeneous tissue- and cell specific plant samples. The employment of this technique with subsequent protein analysis has thus far not been reported for plant tissues, probably due to the difficulties associated with defining a reasonable cellular morphology and, in parallel, allowing efficient protein extraction from tissue samples. The relatively large sample amount needed for successful proteome analysis is an additional issue that complicates protein profiling on a tissue- or even cell-specific level. In contrast to transcript profiling that can be performed from very small sample amounts due to efficient amplification strategies, there is as yet no amplification procedure for proteins available. In the current study, we compared different tissue preparation techniques prior to LM/laser pressure catapulting (LMPC) with respect to their suitability for protein retrieval. Cryosectioning was identified as the best compromise between tissue morphology and effective protein extraction. After collection of vascular bundles from Arabidopsis thaliana stem tissue by LMPC, proteins were extracted and subjected to protein analysis, either by classical two-dimensional gel electrophoresis (2-DE), or by high-efficiency liquid chromatography (LC) in conjunction with tandem mass spectrometry (MS/MS). Our results demonstrate that both methods can be used with LMPC collected plant material. But because of the significantly lower sample amount required for LC-MS/MS than for 2-DE, the combination of LMPC and LC-MS/MS has a higher potential to promote comprehensive proteome analysis of specific plant tissues.

  17. Transgenic Zebrafish Reveal Tissue-Specific Differences in Estrogen Signaling in Response to Environmental Water Samples

    PubMed Central

    Iwanowicz, Luke R.; Hung, Alice L.; Blazer, Vicki S.; Halpern, Marnie E.

    2014-01-01

    Background: Environmental endocrine disruptors (EEDs) are exogenous chemicals that mimic endogenous hormones such as estrogens. Previous studies using a zebrafish transgenic reporter demonstrated that the EEDs bisphenol A and genistein preferentially activate estrogen receptors (ERs) in the larval heart compared with the liver. However, it was not known whether the transgenic zebrafish reporter was sensitive enough to detect estrogens from environmental samples, whether environmental estrogens would exhibit tissue-specific effects similar to those of BPA and genistein, or why some compounds preferentially target receptors in the heart. Methods: We tested surface water samples using a transgenic zebrafish reporter with tandem estrogen response elements driving green fluorescent protein expression (5xERE:GFP). Reporter activation was colocalized with tissue-specific expression of ER genes by RNA in situ hybridization. Results: We observed selective patterns of ER activation in transgenic fish exposed to river water samples from the Mid-Atlantic United States, with several samples preferentially activating receptors in embryonic and larval heart valves. We discovered that tissue specificity in ER activation was due to differences in the expression of ER subtypes. ERα was expressed in developing heart valves but not in the liver, whereas ERβ2 had the opposite profile. Accordingly, subtype-specific ER agonists activated the reporter in either the heart valves or the liver. Conclusion: The use of 5xERE:GFP transgenic zebrafish revealed an unexpected tissue-specific difference in the response to environmentally relevant estrogenic compounds. Exposure to estrogenic EEDs in utero was associated with adverse health effects, with the potentially unanticipated consequence of targeting developing heart valves. Citation: Gorelick DA, Iwanowicz LR, Hung AL, Blazer VS, Halpern ME. 2014. Transgenic zebrafish reveal tissue-specific differences in estrogen signaling in response to

  18. Determination of metronidazole residues in water, sediment and fish tissue samples.

    PubMed

    Wagil, Marta; Maszkowska, Joanna; Białk-Bielińska, Anna; Caban, Magda; Stepnowski, Piotr; Kumirska, Jolanta

    2015-01-01

    Metronidazole (MNZ) is an antibacterial and antiprotozoal drug used in veterinary and human medicine. Its continual entry into the environment and its biological properties may have significant, long-term effects on the stability of ecosystems because MNZ and its metabolites possess mutagenic, carcinogenic and toxic properties. For this reason, the application of MNZ in food-producing species is prohibited in the EU, the USA and other countries. To ensure human food safety and to protect the environment, robust and reliable screening and confirmatory tests capable of the low-level detection of MNZ residues are required. The development of methods for MNZ determination in biological and environmental samples is thus an important analytical task in environmental and food science. This work focuses on the evaluation of a method for determining MNZ in water, sediment and fish tissue samples using liquid chromatography--ion trap mass spectrometry (LC-MS/MS). MNZ was extracted from waters on Strata XC cartridges using solid phase extraction (SPE), and from sediments and fish tissues by solid-liquid extraction (sediment: 15 mL 0.1 M HCl (pH=0.6), 15 min; fish tissue: 15 mL 1% CH3COOH in ACN, 1 min; drying: 5 g MgSO4(anhyd.; 30 s) with SPE purification of the extracts (from sediment: Strata XC cartridge; from fish tissue: Supelco NH2 cartridge). The optimal procedure that we developed was validated in order to confirm its reliability and sensitivity. Matrix effects (ME) were established. Absolute recoveries ranged from 89.3% to 97.2%, and the method detection limits were 3.4 ng L(-1) (water samples), 0.4 ng g(-1) (sediment samples) and 0.3 ng g(-1) (tissue samples). These methods were used to determine MNZ in surface waters, sediments and fish tissues from the Polish River Gościcina; MNZ was found in all these matrices. The highest concentrations in water, sediment and tissue were 136.2 ng L(-1), 12.0 ng g(-1) and 1.5 ng g(-1) respectively. The results confirmed that

  19. The Novel Application of Non-Lethal Citizen Science Tissue Sampling in Recreational Fisheries

    PubMed Central

    Williams, Samuel M.; Holmes, Bonnie J.; Pepperell, Julian G.

    2015-01-01

    Increasing fishing pressure and uncertainty surrounding recreational fishing catch and effort data promoted the development of alternative methods for conducting fisheries research. A pilot investigation was undertaken to engage the Australian game fishing community and promote the non-lethal collection of tissue samples from the black marlin Istiompax indica, a valuable recreational-only species in Australian waters, for the purpose of future genetic research. Recruitment of recreational anglers was achieved by publicizing the project in magazines, local newspapers, social media, blogs, websites and direct communication workshops at game fishing tournaments. The Game Fishing Association of Australia and the Queensland Game Fishing Association were also engaged to advertise the project and recruit participants with a focus on those anglers already involved in the tag-and-release of marlin. Participants of the program took small tissue samples using non-lethal methods which were stored for future genetic analysis. The program resulted in 165 samples from 49 participants across the known distribution of I. indica within Australian waters which was a sufficient number to facilitate a downstream population genetic analysis. The project demonstrated the potential for the development of citizen science sampling programs to collect tissue samples using non-lethal methods in order to achieve targeted research objects in recreationally caught species. PMID:26376487

  20. The Novel Application of Non-Lethal Citizen Science Tissue Sampling in Recreational Fisheries.

    PubMed

    Williams, Samuel M; Holmes, Bonnie J; Pepperell, Julian G

    2015-01-01

    Increasing fishing pressure and uncertainty surrounding recreational fishing catch and effort data promoted the development of alternative methods for conducting fisheries research. A pilot investigation was undertaken to engage the Australian game fishing community and promote the non-lethal collection of tissue samples from the black marlin Istiompax indica, a valuable recreational-only species in Australian waters, for the purpose of future genetic research. Recruitment of recreational anglers was achieved by publicizing the project in magazines, local newspapers, social media, blogs, websites and direct communication workshops at game fishing tournaments. The Game Fishing Association of Australia and the Queensland Game Fishing Association were also engaged to advertise the project and recruit participants with a focus on those anglers already involved in the tag-and-release of marlin. Participants of the program took small tissue samples using non-lethal methods which were stored for future genetic analysis. The program resulted in 165 samples from 49 participants across the known distribution of I. indica within Australian waters which was a sufficient number to facilitate a downstream population genetic analysis. The project demonstrated the potential for the development of citizen science sampling programs to collect tissue samples using non-lethal methods in order to achieve targeted research objects in recreationally caught species.

  1. Non-lethal sampling of walleye for stable isotope analysis: a comparison of three tissues

    USGS Publications Warehouse

    Chipps, Steven R.; VanDeHey, J.A.; Fincel, M.J.

    2012-01-01

    Stable isotope analysis of fishes is often performed using muscle or organ tissues that require sacrificing animals. Non-lethal sampling provides an alternative for evaluating isotopic composition for species of concern or individuals of exceptional value. Stable isotope values of white muscle (lethal) were compared with those from fins and scales (non-lethal) in walleye, Sander vitreus (Mitchill), from multiple systems, size classes and across a range of isotopic values. Isotopic variability was also compared among populations to determine the potential of non-lethal tissues for diet-variability analyses. Muscle-derived isotope values were enriched compared with fins and depleted relative to scales. A split-sample validation technique and linear regression found that isotopic composition of walleye fins and scales was significantly related to that in muscle tissue for both δ13C and δ15N (r2 = 0.79–0.93). However, isotopic variability was significantly different between tissue types in two of six populations for δ15N and three of six populations for δ13C. Although species and population specific, these findings indicate that isotopic measures obtained from non-lethal tissues are indicative of those obtained from muscle.

  2. A new liquid chromatography-tandem mass spectrometry method for determination of parabens in human placental tissue samples.

    PubMed

    Jiménez-Díaz, I; Vela-Soria, F; Zafra-Gómez, A; Navalón, A; Ballesteros, O; Navea, N; Fernández, M F; Olea, N; Vílchez, J L

    2011-05-15

    Endocrine disruptors are a group of organic compounds widely used, which are ubiquitous in the environment and in biological samples. The main effect of these compounds is associated with their ability to mimic or block the action of natural hormones in living organisms, including humans. Parabens (esters of p-hydroxybenzoic acid) belong to this group of compounds. In this work, we propose a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to asses the presence of parabens most commonly used in industrial applications (methyl-, ethyl-, propyl- and butyl-paraben) in samples of human placental tissue. The method involves the extraction of the analytes from the samples using ethyl acetate, followed by a clean-up step using centrifugation prior to their quantification by LC-MS/MS using an atmospheric pressure chemical ionization (APCI) interface in the negative mode. Deuterated bisphenol A (BPA-d(16)) was used as surrogate. Found detection limits (LOD) ranged from 0.03 to 0.06 ng g(-1) and quantification limits (LOQ) from 0.1 to 0.2 ng g(-1), while inter- and intra-day variability was under 13.8%. The method was validated using standard addition calibration and a spike recovery assay. Recovery rates for spiked samples ranged from 82% to 108%. This method was satisfactorily applied for the determination of parabens in 50 placental tissue samples collected from women who live in the province of Granada (Spain).

  3. The inter-sample structural variability of regular tissue-engineered scaffolds significantly affects the micromechanical local cell environment

    PubMed Central

    Campos Marin, A.; Lacroix, D.

    2015-01-01

    Rapid prototyping techniques have been widely used in tissue engineering to fabricate scaffolds with controlled architecture. Despite the ability of these techniques to fabricate regular structures, the consistency with which these regular structures are produced throughout the scaffold and from one scaffold to another needs to be quantified. Small variations at the pore level can affect the local mechanical stimuli sensed by the cells thereby affecting the final tissue properties. Most studies assume rapid prototyping scaffolds as regular structures without quantifying the local mechanical stimuli at the cell level. In this study, a computational method using a micro-computed tomography-based scaffold geometry was developed to characterize the mechanical stimuli within a real scaffold at the pore level. Five samples from a commercial polycaprolactone scaffold were analysed and computational fluid dynamics analyses were created to compare local velocity and shear stress values at the same scaffold location. The five samples did not replicate the computer-aided design (CAD) scaffold and velocity and shear stress values were up to five times higher than the ones calculated in the CAD scaffold. In addition high variability among samples was found: at the same location velocity and shear stress values could be up to two times higher from sample to sample. This study shows that regular scaffolds need to be thoroughly analysed in order to quantify real cell mechanical stimuli so inspection methods should be included as part of the fabrication process. PMID:25844157

  4. Metabolite Profiling of Preneoplastic and Neoplastic Lesions of Oral Cavity Tissue Samples Revealed a Biomarker Pattern

    PubMed Central

    Musharraf, Syed Ghulam; Shahid, Najia; Naqvi, Syed Muhammad Ali; Saleem, Mahwish; Siddiqui, Amna Jabbar; Ali, Anwar

    2016-01-01

    Oral cancer is a major health challenge in the Indian subcontinent and a dreadful form of cancers worldwide. The current study is focused on the identification of distinguished metabolites of oral cancer tissue samples in comparison with precancerous and control tissue samples using gas chromatography coupled with triple quadrupole tandem mass spectrometry and chemometric analyses. Metabolites obtained were identified through National Institute of Standards and Technology (NIST) mass spectral (Wiley registry) library. Mass Profiler Professional (MPP) software was used for the alignment and for all the statistical analysis. 31 compounds out of 735 found distinguishing among oral cancer, precancerous and control group samples using p-value ≤ 0.05. Partial Least Square Discriminant Analysis (PLSDA) model was generated using statistically significant metabolites gave an overall accuracy of 90.2%. Down-regulated amino acid levels appear to be the result of enhanced energy metabolism or up-regulation of the appropriate biosynthetic pathways, and required cell proliferation in cancer tissues. These results suggest that tissue metabolic profiles have great potential in detecting oral cancer and may aid in understanding its underlying mechanisms. PMID:27958349

  5. Facial soft tissue thickness in a sample of Sudanese adults with different occlusions.

    PubMed

    Hamid, Sama; Abuaffan, Amal H

    2016-09-01

    Facial soft tissue thickness is essential to orthodontists and plastic surgeons for treatment planning, and to forensic anthropologists for facial reconstruction, a process combining science and art to recreate a recognizable face from an unidentified skull. The facial profile, together with the age and sex of a person, is related to facial soft tissue thickness, which is required for accurate facial reconstruction and recognition. Skeletal occlusions in orthodontics are classified according to the basic human facial profiles: straight, convex, and concave or skeletal class I, II, and III, respectively. In the present study, the facial soft tissue thickness of 233 Sudanese subjects (105 men and 128 women), ranging in age from 18 to 35 years, with different facial profiles at 20 landmarks was measured (10 soft tissue and 10 dentoskeletal). Sexual dimorphism was noted, with males having thicker facial soft tissue at all measured points. The facial soft tissue thickness varied among different occlusions. Individuals with skeletal class II occlusion had the thickest lower lip, and class III individuals had the thickest upper lip. In general, the Sudanese sample had a unique spectrum of measurements, with thick upper and lower lips, compared with African and Caucasoid subjects, pointing to the need for ethnic-specific data.

  6. Spatially-Resolved Proteomics: Rapid Quantitative Analysis of Laser Capture Microdissected Alveolar Tissue Samples

    SciTech Connect

    Clair, Geremy; Piehowski, Paul D.; Nicola, Teodora; Kitzmiller, Joseph A.; Huang, Eric L.; Zink, Erika M.; Sontag, Ryan L.; Orton, Daniel J.; Moore, Ronald J.; Carson, James P.; Smith, Richard D.; Whitsett, Jeffrey A.; Corley, Richard A.; Ambalavanan, Namasivayam; Ansong, Charles

    2016-12-22

    Global proteomics approaches allow characterization of whole tissue lysates to an impressive depth. However, it is now increasingly recognized that to better understand the complexity of multicellular organisms, global protein profiling of specific spatially defined regions/substructures of tissues (i.e. spatially-resolved proteomics) is essential. Laser capture microdissection (LCM) enables microscopic isolation of defined regions of tissues preserving crucial spatial information. However, current proteomics workflows entail several manual sample preparation steps and are challenged by the microscopic mass-limited samples generated by LCM, and that impact measurement robustness, quantification, and throughput. Here, we coupled LCM with a fully automated sample preparation workflow that with a single manual step allows: protein extraction, tryptic digestion, peptide cleanup and LC-MS/MS analysis of proteomes from microdissected tissues. Benchmarking against the current state of the art in ultrasensitive global proteomic analysis, our approach demonstrated significant improvements in quantification and throughput. Using our LCM-SNaPP proteomics approach, we characterized to a depth of more than 3,400 proteins, the ontogeny of protein changes during normal lung development in laser capture microdissected alveolar tissue containing ~4,000 cells per sample. Importantly, the data revealed quantitative changes for 350 low abundance transcription factors and signaling molecules, confirming earlier transcript-level observations and defining seven modules of coordinated transcription factor/signaling molecule expression patterns, suggesting that a complex network of temporal regulatory control directs normal lung development with epigenetic regulation fine-tuning pre-natal developmental processes. Our LCM-proteomics approach facilitates efficient, spatially-resolved, ultrasensitive global proteomics analyses in high-throughput that will be enabling for several clinical and

  7. Tabletop magnetic resonance elastography for the measurement of viscoelastic parameters of small tissue samples

    NASA Astrophysics Data System (ADS)

    Ipek-Ugay, Selcan; Drießle, Toni; Ledwig, Michael; Guo, Jing; Hirsch, Sebastian; Sack, Ingolf; Braun, Jürgen

    2015-02-01

    We demonstrate the feasibility of low-cost tabletop MR elastography (MRE) for quantifying the complex shear modulus G∗ of small soft biological tissue samples as provided by pathologists. The MRE system was developed based on a tabletop MRI scanner equipped with a 0.5 T permanent magnet and a tissue sample holder mounted to a loudspeaker. A spin echo sequence was enhanced with motion-encoding gradients of 250 mT/m amplitude synchronized to acoustic vibration frequencies. Shear wave images suitable for elastography were acquired between vibration frequencies of 0.5 and 1 kHz in agarose, ultrasound gel, porcine liver, porcine skeletal muscle, and bovine heart with a spatial resolution of 234 μm pixel edge length. The measured frequency dependence of G∗ agreed well with previous work based on high-field MR systems. The ratio between loss and storage moduli was highest in liver and ultrasound gel, followed by muscle tissue and agarose gel while ultrasound gel and liver showed similarly low storage moduli compared to the other samples. The shear wave to noise ratio is an important imaging criteria for MRE and was about 4.2 times lower for the preliminary setup of the 0.5 T tabletop system compared to a 7 T animal scanner. In the future, the new tabletop MRE system may serve as a low cost device for preclinical research on the correlation of viscoelastic parameters with histopathology of biological samples.

  8. Advancements in mass spectrometry for biological samples: Protein chemical cross-linking and metabolite analysis of plant tissues

    SciTech Connect

    Klein, Adam

    2015-01-01

    This thesis presents work on advancements and applications of methodology for the analysis of biological samples using mass spectrometry. Included in this work are improvements to chemical cross-linking mass spectrometry (CXMS) for the study of protein structures and mass spectrometry imaging and quantitative analysis to study plant metabolites. Applications include using matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to further explore metabolic heterogeneity in plant tissues and chemical interactions at the interface between plants and pests. Additional work was focused on developing liquid chromatography-mass spectrometry (LC-MS) methods to investigate metabolites associated with plant-pest interactions.

  9. Targeted or whole genome sequencing of formalin fixed tissue samples: potential applications in cancer genomics.

    PubMed

    Munchel, Sarah; Hoang, Yen; Zhao, Yue; Cottrell, Joseph; Klotzle, Brandy; Godwin, Andrew K; Koestler, Devin; Beyerlein, Peter; Fan, Jian-Bing; Bibikova, Marina; Chien, Jeremy

    2015-09-22

    Current genomic studies are limited by the poor availability of fresh-frozen tissue samples. Although formalin-fixed diagnostic samples are in abundance, they are seldom used in current genomic studies because of the concern of formalin-fixation artifacts. Better characterization of these artifacts will allow the use of archived clinical specimens in translational and clinical research studies. To provide a systematic analysis of formalin-fixation artifacts on Illumina sequencing, we generated 26 DNA sequencing data sets from 13 pairs of matched formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FF) tissue samples. The results indicate high rate of concordant calls between matched FF/FFPE pairs at reference and variant positions in three commonly used sequencing approaches (whole genome, whole exome, and targeted exon sequencing). Global mismatch rates and C · G > T · A substitutions were comparable between matched FF/FFPE samples, and discordant rates were low (<0.26%) in all samples. Finally, low-pass whole genome sequencing produces similar pattern of copy number alterations between FF/FFPE pairs. The results from our studies suggest the potential use of diagnostic FFPE samples for cancer genomic studies to characterize and catalog variations in cancer genomes.

  10. Simultaneous sampling of tissue oxygenation and oxygen consumption in skeletal muscle.

    PubMed

    Nugent, William H; Song, Bjorn K; Pittman, Roland N; Golub, Aleksander S

    2016-05-01

    Under physiologic conditions, microvascular oxygen delivery appears to be well matched to oxygen consumption in respiring tissues. We present a technique to measure interstitial oxygen tension (PISFO2) and oxygen consumption (VO2) under steady-state conditions, as well as during the transitions from rest to activity and back. Phosphorescence Quenching Microscopy (PQM) was employed with pneumatic compression cycling to achieve 1 to 10 Hz sampling rates of interstitial PO2 and simultaneous recurrent sampling of VO2 (3/min) in the exteriorized rat spinotrapezius muscle. The compression pressure was optimized to 120-130 mmHg without adverse effect on the tissue preparation. A cycle of 5s compression followed by 15s recovery yielded a resting VO2 of 0.98 ± 0.03 ml O2/100 cm(3)min while preserving microvascular oxygen delivery. The measurement system was then used to assess VO2 dependence on PISFO2 at rest and further tested under conditions of isometric muscle contraction to demonstrate a robust ability to monitor the on-kinetics of tissue respiration and the compensatory changes in PISFO2 during contraction and recovery. The temporal and spatial resolution of this approach is well suited to studies seeking to characterize microvascular oxygen supply and demand in thin tissues.

  11. Identification of organ tissue types and skin from forensic samples by microRNA expression analysis.

    PubMed

    Sauer, Eva; Extra, Antje; Cachée, Philipp; Courts, Cornelius

    2017-05-01

    The identification of organ tissues in traces recovered from scenes and objects with regard to violent crimes involving serious injuries can be of considerable relevance in forensic investigations. Molecular genetic approaches are provably superior to histological and immunological assays in characterizing organ tissues, and micro-RNAs (miRNAs), due to their cell type specific expression patterns and stability against degradation, emerged as a promising molecular species for forensic analyses, with a range of tried and tested indicative markers. Thus, herein we present the first miRNA based approach for the forensic identification of organ tissues. Using quantitative PCR employing an empirically derived strategy for data normalization and unbiased statistical decision making, we assessed the differential expression of 15 preselected miRNAs in tissues of brain, kidney, lung, liver, heart muscle, skeletal muscle and skin. We show that not only can miRNA expression profiling be used to reliably differentiate between organ tissues but also that this method, which is compatible with and complementary to forensic DNA analysis, is applicable to realistic forensic samples e.g. mixtures, aged and degraded material as well as traces generated by mock stabbings and experimental shootings at ballistic models.

  12. Effects of sample preparation on the optical properties of breast tissue

    NASA Astrophysics Data System (ADS)

    Marks, Fay A.

    1996-04-01

    The optical properties of biological tissue should be determined in vivo whenever possible. However, for those instances when in vivo studies are impractical, too expensive or inappropriate, and when blood flow is not an issue, the ability to perform in vitro studies then becomes invaluable. Optical absorption spectroscopy shows that it may be possible to obtain meaningful information about the optical properties of human breast tissue from in vitro samples if strict preparation and measuring protocols are used. That a strict protocol for storing and handling tissue is critical can be seen from our observations of changes in the optical absorption spectra that occur in response to formalin fixation, the passage of time, application of stains and dyes, and storage in growth medium of the excised tissue. In vivo optical absorption spectroscopy measurements have been made on human breast cancer xenografts and compared with in vitro measurements on breast biopsies prepared according to precise collection and treatment protocols. There is a 'window of opportunity' before time dependent changes in the UV optical absorption spectra of the excised tissue specimens occur. This time window of opportunity widens at longer wavelengths with the least changes occurring in the optical spectra in the NIR.

  13. Transgenic zebrafish reveal tissue-specific differences in estrogen signaling in response to environmental water samples

    USGS Publications Warehouse

    Gorelick, Daniel A.; Iwanowicz, Luke R.; Hung, Alice L.; Blazer, Vicki; Halpern, Marnie E.

    2014-01-01

    Background: Environmental endocrine disruptors (EED) are exogenous chemicals that mimic endogenous hormones, such as estrogens. Previous studies using a zebrafish transgenic reporter demonstrated that the EEDs bisphenol A and genistein preferentially activate estrogen receptors (ER) in the larval heart compared to the liver. However, it was not known whether the transgenic zebrafish reporter was sensitive enough to detect estrogens from environmental samples, whether environmental estrogens would exhibit similar tissue-specific effects as BPA and genistein or why some compounds preferentially target receptors in the heart. Methods: We tested surface water samples using a transgenic zebrafish reporter with tandem estrogen response elements driving green fluorescent protein expression (5xERE:GFP). Reporter activation was colocalized with tissue-specific expression of estrogen receptor genes by RNA in situ hybridization. Results: Selective patterns of ER activation were observed in transgenic fish exposed to river water samples from the Mid-Atlantic United States, with several samples preferentially activating receptors in embryonic and larval heart valves. We discovered that tissue-specificity in ER activation is due to differences in the expression of estrogen receptor subtypes. ERα is expressed in developing heart valves but not in the liver, whereas ERβ2 has the opposite profile. Accordingly, subtype-specific ER agonists activate the reporter in either the heart valves or the liver. Conclusion: The use of 5xERE:GFP transgenic zebrafish has revealed an unexpected tissue-specific difference in the response to environmentally relevant estrogenic compounds. Exposure to estrogenic EEDs in utero is associated with adverse health effects, with the potentially unanticipated consequence of targeting developing heart valves.

  14. Proteogenomic Analysis of Human Chromosome 9-Encoded Genes from Human Samples and Lung Cancer Tissues

    PubMed Central

    Ahn, Jung-Mo; Kim, Min-Sik; Kim, Yong-In; Jeong, Seul-Ki; Lee, Hyoung-Joo; Lee, Sun Hee; Paik, Young-Ki; Pandey, Akhilesh; Cho, Je-Yoel

    2014-01-01

    The Chromosome-centric Human Proteome Project (C-HPP) was recently initiated as an international collaborative effort. Our team adopted chromosome 9 (Chr 9) and performed a bioinformatics and proteogenomic analysis to catalog Chr 9-encoded proteins from normal tissues, lung cancer cell lines and lung cancer tissues. Approximately 74.7% of the Chr 9 genes of the human genome were identified, which included approximately 28% of missing proteins (46 of 162) on Chr 9 compared with the list of missing proteins from the neXtProt master table (2013-09). In addition, we performed a comparative proteomics analysis between normal lung and lung cancer tissues. Based on the data analysis, 15 proteins from Chr 9 were detected only in lung cancer tissues. Finally, we conducted a proteogenomic analysis to discover Chr 9-residing single nucleotide polymorphisms (SNP) and mutations described in the COSMIC cancer mutation database. We identified 21 SNPs and 4 mutations containing peptides on Chr 9 from normal human cells/tissues and lung cancer cell lines, respectively. In summary, this study provides valuable information of the human proteome for the scientific community as part of C-HPP. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD. PMID:24274035

  15. Evidence That Certain Waste Tank Headspace Vapor Samples Were Contaminated by Semivolatile Polymer Additives

    SciTech Connect

    Huckaby, James L.

    2006-02-09

    Vapor samples collected from the headspaces of the Hanford Site high-level radioactive waste tanks in 1994 and 1995 using the Vapor Sampling System (VSS) were reported to contain trace levels of phthalates, antioxidants, and certain other industrial chemicals that did not have a logical origin in the waste. This report examines the evidence these chemicals were sampling artifacts (contamination) and identifies the chemicals reported as headspace constituents that may instead have been contaminants. Specific recommendations are given regarding the marking of certain chemicals as suspect on the basis they were sampling manifold contaminants.

  16. Sensitive PCR analysis of animal tissue samples for fragments of endogenous and transgenic plant DNA.

    PubMed

    Nemeth, Anne; Wurz, Andreas; Artim, Lori; Charlton, Stacy; Dana, Greg; Glenn, Kevin; Hunst, Penny; Jennings, James; Shilito, Ray; Song, Ping

    2004-10-06

    An optimized DNA extraction protocol for animal tissues coupled with sensitive PCR methods was used to determine whether trace levels of feed-derived DNA fragments, plant and/or transgenic, are detectable in animal tissue samples including dairy milk and samples of muscle (meat) from chickens, swine, and beef steers. Assays were developed to detect DNA fragments of both the high copy number chloroplast-encoded maize rubisco gene (rbcL) and single copy nuclear-encoded transgenic elements (p35S and a MON 810-specific gene fragment). The specificities of the two rbcL PCR assays and two transgenic DNA PCR assays were established by testing against a range of conventional plant species and genetically modified maize crops. The sensitivities of the two rbcL PCR assays (resulting in 173 and 500 bp amplicons) were similar, detecting as little as 0.08 and 0.02 genomic equivalents, respectively. The sensitivities of the p35S and MON 810 PCR assays were approximately 5 and 10 genomic equivalents for 123 bp and 149 bp amplicons, respectively, which were considerably less than the sensitivity of the rbcL assays in terms of plant cell equivalents, but approximately similar when the higher numbers of copies of the chloroplast genome per cell are taken into account. The 173 bp rbcL assay detected the target plant chloroplast DNA fragment in 5%, 15%, and 53% of the muscle samples from beef steers, broiler chickens, and swine, respectively, and in 86% of the milk samples from dairy cows. Reanalysis of new aliquots of 31 of the pork samples that were positive in the 173 bp rbcL PCR showed that 58% of these samples were reproducibly positive in this same PCR assay. The 500 bp rbcL assay detected DNA fragments in 43% of the swine muscle samples and 79% of the milk samples. By comparison, no statistically significant detections of transgenic DNA fragments by the p35S PCR assay occurred with any of these animal tissue samples.

  17. Threshold-dependent sample sizes for selenium assessment with stream fish tissue

    USGS Publications Warehouse

    Hitt, Nathaniel P.; Smith, David R.

    2015-01-01

    Natural resource managers are developing assessments of selenium (Se) contamination in freshwater ecosystems based on fish tissue concentrations. We evaluated the effects of sample size (i.e., number of fish per site) on the probability of correctly detecting mean whole-body Se values above a range of potential management thresholds. We modeled Se concentrations as gamma distributions with shape and scale parameters fitting an empirical mean-to-variance relationship in data from southwestern West Virginia, USA (63 collections, 382 individuals). We used parametric bootstrapping techniques to calculate statistical power as the probability of detecting true mean concentrations up to 3 mg Se/kg above management thresholds ranging from 4 to 8 mg Se/kg. Sample sizes required to achieve 80% power varied as a function of management thresholds and Type I error tolerance (α). Higher thresholds required more samples than lower thresholds because populations were more heterogeneous at higher mean Se levels. For instance, to assess a management threshold of 4 mg Se/kg, a sample of eight fish could detect an increase of approximately 1 mg Se/kg with 80% power (given α = 0.05), but this sample size would be unable to detect such an increase from a management threshold of 8 mg Se/kg with more than a coin-flip probability. Increasing α decreased sample size requirements to detect above-threshold mean Se concentrations with 80% power. For instance, at an α-level of 0.05, an 8-fish sample could detect an increase of approximately 2 units above a threshold of 8 mg Se/kg with 80% power, but when α was relaxed to 0.2, this sample size was more sensitive to increasing mean Se concentrations, allowing detection of an increase of approximately 1.2 units with equivalent power. Combining individuals into 2- and 4-fish composite samples for laboratory analysis did not decrease power because the reduced number of laboratory samples was compensated for by increased

  18. The relationship between decorrelation time and sample thickness in acute rat brain tissue slices (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Brake, Joshua; Jang, Mooseok; Yang, Changhuei

    2016-03-01

    The optical opacity of biological tissue has long been a challenge in biomedical optics due to the strong scattering nature of tissue in the optical regime. While most conventional optical techniques attempt to gate out multiply scattered light and use only unscattered light, new approaches in the field of wavefront shaping exploit the time reversible symmetry of optical scattering in order to focus light inside or through scattering media. While these approaches have been demonstrated effectively on static samples, it has proven difficult to apply them to dynamic biological samples since even small changes in the relative positions of the scatterers within will cause the time symmetry that wavefront shaping relies upon to decorrelate. In this paper we investigate the decorrelation curves of acute rat brain slices for thicknesses in the range 1-3 mm (1/e decorrelation time on the order of seconds) using multi-speckle diffusing wave spectroscopy (MSDWS) and compare the results with theoretical predictions. The results of this study demonstrate that the 1/L^2 relationship between decorrelation time and thickness predicted by diffusing wave spectroscopy provides a good rule of thumb for estimating how the decorrelation of a sample will change with increasing thickness. Understanding this relationship will provide insight to guide the future development of biophotonic wavefront shaping tools by giving an estimate of how fast wavefront shaping systems need to operate to overcome the dynamic nature of biological samples.

  19. A single lysis solution for the analysis of tissue samples by different proteomic technologies.

    PubMed

    Gromov, Pavel; Celis, Julio E; Gromova, Irina; Rank, Fritz; Timmermans-Wielenga, Vera; Moreira, José M A

    2008-12-01

    Cancer, being a major healthcare concern worldwide, is one of the main targets for the application of emerging proteomic technologies and these tools promise to revolutionize the way cancer will be diagnosed and treated in the near future. Today, as a result of the unprecedented advances that have taken place in molecular biology, cell biology and genomics there is a pressing need to accelerate the translation of basic discoveries into clinical applications. This need, compounded by mounting evidence that cellular model systems are unable to fully recapitulate all biological aspects of human dissease, is driving scientists to increasingly use clinically relevant samples for biomarker and target discovery. Tissues are heterogeneous and as a result optimization of sample preparation is critical for generating accurate, representative, and highly reproducible quantitative data. Although a large number of protocols for preparation of tissue lysates has been published, so far no single recipe is able to provide a "one-size fits all" solubilization procedure that can be used to analyse the same lysate using different proteomics technologies. Here we present evidence showing that cell lysis buffer 1 (CLB1), a lysis solution commercialized by Zeptosens [a division of Bayer (Schweiz) AG], provides excellent sample solubilization and very high 2D PAGE protein resolution both when using carrier ampholytes and immobilized pH gradient strips. Moreover, this buffer can also be used for array-based proteomics (reverse-phase lysate arrays or direct antibody arrays), allowing the direct comparison of qualitative and quantitative data yielded by these technologies when applied to the same samples. The usefulness of the CLB1 solution for gel-based proteomics was further established by 2D PAGE analysis of a number of technically demanding specimens such as breast carcinoma core needle biopsies and problematic tissues such as brain cortex, cerebellum, skeletal muscle, kidney cortex and

  20. Determination of optical properties of oxidative bleaching human dental tissue samples using optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Ni, Y. R.; Guo, Z. Y.; Shu, S. Y.; Zeng, C. C.; Zhong, H. Q.; Chen, B. L.; Liu, Z. M.; Bao, Y.

    2011-10-01

    Oxidative bleaching changes of human teeth induced changes in the optical properties of dental tissue. We introduced 1310 nm wavelengths of optical coherence tomography (OCT) attenuation coefficient method which is a relatively novel and rarely reported methodology to measure the correlation coefficient during the teeth oxidative bleaching procedure. And the quantitative parameters of enamel optical thickness and disruption of the entrance signal (DES) were extracted from the OCT images. The attenuation coefficient of the bleached tissue is 6.2 mm-1 which is significant (p < 0.001) higher than that unbleached sample is 1.4 mm-1. But attenuation coefficient varied significantly (p < 0.001) between 5.9 and 1.5 mm-1 in dentine which is downtrend. Furthermore, the persistence of bleaching oxidation in 35% hydrogen peroxide-induced optical thickness of enamel is similar with unbleached tissue which may indicate the refractive index of enamel is unchanged. Moreover, disruption of the entrance signal (DES) analysis showed that remarkable difference was appeared at enamel surface. The results indicate that optical properties of oxidative bleaching human dental tissue can be determined by attenuation coefficient using OCT system.

  1. Spatially-Resolved Proteomics: Rapid Quantitative Analysis of Laser Capture Microdissected Alveolar Tissue Samples.

    PubMed

    Clair, Geremy; Piehowski, Paul D; Nicola, Teodora; Kitzmiller, Joseph A; Huang, Eric L; Zink, Erika M; Sontag, Ryan L; Orton, Daniel J; Moore, Ronald J; Carson, James P; Smith, Richard D; Whitsett, Jeffrey A; Corley, Richard A; Ambalavanan, Namasivayam; Ansong, Charles

    2016-12-22

    Laser capture microdissection (LCM)-enabled region-specific tissue analyses are critical to better understand complex multicellular processes. However, current proteomics workflows entail several manual sample preparation steps and are challenged by the microscopic mass-limited samples generated by LCM, impacting measurement robustness, quantification and throughput. Here, we coupled LCM with a proteomics workflow that provides fully automated analysis of proteomes from microdissected tissues. Benchmarking against the current state-of-the-art in ultrasensitive global proteomics (FASP workflow), our approach demonstrated significant improvements in quantification (~2-fold lower variance) and throughput (>5 times faster). Using our approach we for the first time characterized, to a depth of >3,400 proteins, the ontogeny of protein changes during normal lung development in microdissected alveolar tissue containing only 4,000 cells. Our analysis revealed seven defined modules of coordinated transcription factor-signaling molecule expression patterns, suggesting a complex network of temporal regulatory control directs normal lung development with epigenetic regulation fine-tuning pre-natal developmental processes.

  2. Spatially-Resolved Proteomics: Rapid Quantitative Analysis of Laser Capture Microdissected Alveolar Tissue Samples

    PubMed Central

    Clair, Geremy; Piehowski, Paul D.; Nicola, Teodora; Kitzmiller, Joseph A.; Huang, Eric L.; Zink, Erika M.; Sontag, Ryan L.; Orton, Daniel J.; Moore, Ronald J.; Carson, James P.; Smith, Richard D.; Whitsett, Jeffrey A.; Corley, Richard A.; Ambalavanan, Namasivayam; Ansong, Charles

    2016-01-01

    Laser capture microdissection (LCM)-enabled region-specific tissue analyses are critical to better understand complex multicellular processes. However, current proteomics workflows entail several manual sample preparation steps and are challenged by the microscopic mass-limited samples generated by LCM, impacting measurement robustness, quantification and throughput. Here, we coupled LCM with a proteomics workflow that provides fully automated analysis of proteomes from microdissected tissues. Benchmarking against the current state-of-the-art in ultrasensitive global proteomics (FASP workflow), our approach demonstrated significant improvements in quantification (~2-fold lower variance) and throughput (>5 times faster). Using our approach we for the first time characterized, to a depth of >3,400 proteins, the ontogeny of protein changes during normal lung development in microdissected alveolar tissue containing only 4,000 cells. Our analysis revealed seven defined modules of coordinated transcription factor-signaling molecule expression patterns, suggesting a complex network of temporal regulatory control directs normal lung development with epigenetic regulation fine-tuning pre-natal developmental processes. PMID:28004771

  3. Molecular strain identification of the Mycobacterium tuberculosis complex in archival tissue samples

    PubMed Central

    Zink, A R; Nerlich, A G

    2004-01-01

    Aims: To investigate the use of different molecular analyses that can identify distinct strains of human pathogenic mycobacteria in formalin fixed and paraffin wax embedded archival tissue samples to see whether it is possible to differentiate between the members of the Mycobacterium tuberculosis complex (M tuberculosis, M bovis, M africanum, M microti, or M canettii) and/or substrains in a high number of samples. This would be of interest for identifying individual infection traits and superinfection by different mycobacterial strains. Methods: Forty nine archival tissue samples with clinically and/or histologically suspected tuberculosis infection were subjected to molecular DNA analysis. Results: The molecular analysis revealed the presence of M tuberculosis complex DNA in 20 samples, whereas acid fast bacilli could be detected by Ziehl-Neelsen staining in only eight samples. All IS6110 positive samples were further characterised by spoligotyping and seven cases provided M tuberculosis specific signatures, whereas M bovis specific signatures were obtained in four cases. The analysis of mtp40, oxyR, and pncA partial gene sequences confirmed the presence of M tuberculosis in six cases and M bovis in one case. The amplification and sequencing of four further genetic regions (katG, gyrA, TbD1, RD9) characterised six “modern” M tuberculosis strains belonging to genetic groups 2 or 3. Conclusion: This study provides clear evidence that archival paraffin wax embedded material can be used for further studies on the strain identification of M tuberculosis complex strains and can therefore unequivocally be used for the study of the epidemiology and evolution of tuberculosis pathogens. PMID:15509681

  4. Risk for molecular contamination of tissue samples evaluated for targeted anti-cancer therapy

    PubMed Central

    Simon, Einav; Fahoum, Ibrahim; Sabo, Edmond; Ben-Izhak, Ofer; Hershkovitz, Dov

    2017-01-01

    With the increasing usage of sensitive PCR technology for pharmacogenetics, cross contamination becomes a significant concern. Researchers employed techniques which basically include replacing laboratory equipment after each sample preparation; however, there are no recommended guidelines. In the present work we wanted to evaluate the risk of cross contamination during tissue processing using the routine precaution measures. Twenty-one surgical samples of lung adenocarcinoma were used, of which 7 contained EGFR exon 19 mutation, 7 contained EGFR exon 21 mutation (p.L858R) and 7 were EGFR wild-type. The samples were ordered by alternating the mutation group to maximize the potential for cross contamination and underwent tissue sectioning and de-paraffinization. The entire process was performed using the same tools. Following DNA extraction all samples underwent PCR amplification and were scrutinized for small fractions of EGFR mutation using deep sequencing with the Ion torrent PGM technology. Twenty samples yielded results. The fraction of mutated copies was 41 ± 23% (range 11–66) for the cases with known exon 19 mutation and 48±24% (range 0–65) for the cases with known exon 21 mutations. No in-frame exon 19 deletion mutations were identified in the wild-type (WT) and exon 21 groups. The fraction of EGFR exon 21 (codon 858) mutations was 0.018±0.014% (range 0–0.05%) in the WT and exon 19 groups, which was not statistically different than the background sequencing artifact noise for the same base-pair alteration (p = 0.21). Our results suggest that standard precautions are sufficient for molecular pathology diagnosis of surgical samples and are not associated with increased risk of cross contamination. PMID:28288198

  5. Indonesia: statistical sampling technique in validation of radiation sterilisation dose of biological tissue.

    PubMed

    Hilmy, N; Basril, A; Febrida, A

    2003-01-01

    The aim of the work is to find the best solution for statistical sampling technique in validation of radiation sterilization dose (RSD) for biological tissues, according to ISO standard. As a model for sampling are biological tissues retrieved from one cadaver donor which consist of frozen bone grafts (18 packets), lyophilized allografts (68 packets) and demineralized bone powder grafts (40 packets). The size and type of products vary from long bones, cancellous chips to bone powders, tendons and facia lata, that make the number of bioburden per product could not be treated equally. Frozen samples could not be considered as the same production batch with lyophilized samples, because of different processing and irradiation temperature. The minimum number of uniformed samples needed for validation per production batch size, according to ISO 13409, is from 20 to 79 and 20 of them will be used for the test sample size, i.e. 10 for bio-burden determination and the remaining 10 for verification dose. Based on the number of uniformed grafts, statistical sampling can be carried out on lyophilized and demineralized bone grafts, but not on frozen bone grafts. Bioburden determinations were carried out and validated according to ISO 11737-1. Results of average bioburden determination (cfu/per packet), using sample item portion (SIP) = 1, are 5 cfu/packet for lyophilized bone grafts and 4 cfu/packet for demineralized bone powder grafts. Verification doses obtained were 2.40 kGy for lyophilized grafts and 2.24 kGy for demineralized bone powder grafts. The results of verification dose were accepted and the RSD of 25 kGy is substantiated It can be concluded that a statistical sampling technique can be applied if all the grafts produced in the same process such as lyophilized, demineralized as well as frozen are assumed to be in one production batch regardless of sample uniformity such as size, type and weight; for this ISO 13409 can be applied for the validation of RSD.

  6. Liquid microjunction surface sampling of acetaminophen, terfenadine and their metabolites in thin tissue sections

    DOE PAGES

    Kertesz, Vilmos; Paranthaman, Nithya; Moench, Paul; ...

    2014-10-01

    The aim of this paper was to evaluate the analytical performance of a fully automated droplet-based surface-sampling system for determining the distribution of the drugs acetaminophen and terfenadine, and their metabolites, in rat thin tissue sections. The following are the results: The rank order of acetaminophen concentration observed in tissues was stomach > small intestine > liver, while the concentrations of its glucuronide and sulfate metabolites were greatest in the liver and small intestine. Terfenadine was most concentrated in the liver and kidney, while its major metabolite, fexofenadine, was found in the liver and small intestine. In conclusion, the spatialmore » distributions of both drugs and their respective metabolites observed in this work were consistent with previous studies using radiolabeled drugs.« less

  7. Liquid microjunction surface sampling of acetaminophen, terfenadine and their metabolites in thin tissue sections

    SciTech Connect

    Kertesz, Vilmos; Paranthaman, Nithya; Moench, Paul; Catoire, Alexandre; Flarakos, Jimmy; Van Berkel, Gary J.

    2014-10-01

    The aim of this paper was to evaluate the analytical performance of a fully automated droplet-based surface-sampling system for determining the distribution of the drugs acetaminophen and terfenadine, and their metabolites, in rat thin tissue sections. The following are the results: The rank order of acetaminophen concentration observed in tissues was stomach > small intestine > liver, while the concentrations of its glucuronide and sulfate metabolites were greatest in the liver and small intestine. Terfenadine was most concentrated in the liver and kidney, while its major metabolite, fexofenadine, was found in the liver and small intestine. In conclusion, the spatial distributions of both drugs and their respective metabolites observed in this work were consistent with previous studies using radiolabeled drugs.

  8. A workflow to preserve genome-quality tissue samples from plants in botanical gardens and arboreta1

    PubMed Central

    Gostel, Morgan R.; Kelloff, Carol; Wallick, Kyle; Funk, Vicki A.

    2016-01-01

    Premise of the study: Internationally, gardens hold diverse living collections that can be preserved for genomic research. Workflows have been developed for genomic tissue sampling in other taxa (e.g., vertebrates), but are inadequate for plants. We outline a workflow for tissue sampling intended for two audiences: botanists interested in genomics research and garden staff who plan to voucher living collections. Methods and Results: Standard herbarium methods are used to collect vouchers, label information and images are entered into a publicly accessible database, and leaf tissue is preserved in silica and liquid nitrogen. A five-step approach for genomic tissue sampling is presented for sampling from living collections according to current best practices. Conclusions: Collecting genome-quality samples from gardens is an economical and rapid way to make available for scientific research tissue from the diversity of plants on Earth. The Global Genome Initiative will facilitate and lead this endeavor through international partnerships. PMID:27672517

  9. Evaluation of sample holders designed for long-lasting X-ray micro-tomographic scans of ex-vivo soft tissue samples

    NASA Astrophysics Data System (ADS)

    Dudak, J.; Zemlicka, J.; Krejci, F.; Karch, J.; Patzelt, M.; Zach, P.; Sykora, V.; Mrzilkova, J.

    2016-03-01

    X-ray microradiography and microtomography are imaging techniques with increasing applicability in the field of biomedical and preclinical research. Application of hybrid pixel detector Timepix enables to obtain very high contrast of low attenuating materials such as soft biological tissue. However X-ray imaging of ex-vivo soft tissue samples is a difficult task due to its structural instability. Ex-vivo biological tissue is prone to fast drying-out which is connected with undesired changes of sample size and shape producing later on artefacts within the tomographic reconstruction. In this work we present the optimization of our Timepix equipped micro-CT system aiming to maintain soft tissue sample in stable condition. Thanks to the suggested approach higher contrast of tomographic reconstructions can be achieved while also large samples that require detector scanning can be easily measured.

  10. Normalization of gene expression measurement of tissue samples obtained by transurethral resection of bladder tumors

    PubMed Central

    Pop, Laura A; Pileczki, Valentina; Cojocneanu-Petric, Roxana M; Petrut, Bogdan; Braicu, Cornelia; Jurj, Ancuta M; Buiga, Rares; Achimas-Cadariu, Patriciu; Berindan-Neagoe, Ioana

    2016-01-01

    Background Sample processing is a crucial step for all types of genomic studies. A major challenge for researchers is to understand and predict how RNA quality affects the identification of transcriptional differences (by introducing either false-positive or false-negative errors). Nanotechnologies help improve the quality and quantity control for gene expression studies. Patients and methods The study was performed on 14 tumor and matched normal pairs of tissue from patients with bladder urothelial carcinomas. We assessed the RNA quantity by using the NanoDrop spectrophotometer and the quality by nano-microfluidic capillary electrophoresis technology provided by Agilent 2100 Bioanalyzer. We evaluated the amplification status of three housekeeping genes and one small nuclear RNA gene using the ViiA 7 platform, with specific primers. Results Every step of the sample handling protocol, which begins with sample harvest and ends with the data analysis, is of utmost importance due to the fact that it is time consuming, labor intensive, and highly expensive. High temperature of the surgical procedure does not affect the small nucleic acid sequences in comparison with the mRNA. Conclusion Gene expression is clearly affected by the RNA quality, but less affected in the case of small nuclear RNAs. We proved that the high-temperature, highly invasive transurethral resection of bladder tumor procedure damages the tissue and affects the integrity of the RNA from biological specimens. PMID:27330317

  11. Histology and Biaxial Mechanical Behavior of Abdominal Aortic Aneurysm Tissue Samples.

    PubMed

    Pancheri, Francesco Q; Peattie, Robert A; Reddy, Nithin D; Ahamed, Touhid; Lin, Wenjian; Ouellette, Timothy D; Iafrati, Mark D; Luis Dorfmann, A

    2017-03-01

    Abdominal aortic aneurysms (AAAs) represent permanent, localized dilations of the abdominal aorta that can be life-threatening if progressing to rupture. Evaluation of risk of rupture depends on understanding the mechanical behavior of patient AAA walls. In this project, a series of patient AAA wall tissue samples have been evaluated through a combined anamnestic, mechanical, and histopathologic approach. Mechanical properties of the samples have been characterized using a novel, strain-controlled, planar biaxial testing protocol emulating the in vivo deformation of the aorta. Histologically, the tissue ultrastructure was highly disrupted. All samples showed pronounced mechanical stiffening with stretch and were notably anisotropic, with greater stiffness in the circumferential than the axial direction. However, there were significant intrapatient variations in wall stiffness and stress. In biaxial tests in which the longitudinal stretch was held constant at 1.1 as the circumferential stretch was extended to 1.1, the maximum average circumferential stress was 330 ± 70 kPa, while the maximum average axial stress was 190 ± 30 kPa. A constitutive model considering the wall as anisotropic with two preferred directions fit the measured data well. No statistically significant differences in tissue mechanical properties were found based on patient gender, age, maximum bulge diameter, height, weight, body mass index, or smoking history. Although a larger patient cohort is merited to confirm these conclusions, the project provides new insight into the relationships between patient natural history, histopathology, and mechanical behavior that may be useful in the development of accurate methods for rupture risk evaluation.

  12. Microscopy and elemental analysis in tissue samples using computed microtomography with synchrotron x-rays

    SciTech Connect

    Spanne, P.; Rivers, M.L.

    1988-01-01

    The initial development shows that CMT using synchrotron x-rays can be developed to ..mu..m spatial resolution and perhaps even better. This creates a new microscopy technique which is of special interest in morphological studies of tissues, since no chemical preparation or slicing of the sample is necessary. The combination of CMT with spatial resolution in the ..mu..m range and elemental mapping with sensitivity in the ppM range results in a new tool for elemental mapping at the cellular level. 7 refs., 1 fig.

  13. 49 CFR 199.111 - Retention of samples and additional testing.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... request, the sample may be discarded following the end of the 365-day period. (b) If the medical review officer (MRO) determines there is no legitimate medical explanation for a confirmed positive test result... and Human Services. The operator may require the employee to pay in advance the cost of shipment...

  14. Additive manufacturing of wet-spun polymeric scaffolds for bone tissue engineering.

    PubMed

    Puppi, Dario; Mota, Carlos; Gazzarri, Matteo; Dinucci, Dinuccio; Gloria, Antonio; Myrzabekova, Mairam; Ambrosio, Luigi; Chiellini, Federica

    2012-12-01

    An Additive Manufacturing technique for the fabrication of three-dimensional polymeric scaffolds, based on wet-spinning of poly(ε-caprolactone) (PCL) or PCL/hydroxyapatite (HA) solutions, was developed. The processing conditions to fabricate scaffolds with a layer-by-layer approach were optimized by studying their influence on fibres morphology and alignment. Two different scaffold architectures were designed and fabricated by tuning inter-fibre distance and fibres staggering. The developed scaffolds showed good reproducibility of the internal architecture characterized by highly porous, aligned fibres with an average diameter in the range 200-250 μm. Mechanical characterization showed that the architecture and HA loading influenced the scaffold compressive modulus and strength. Cell culture experiments employing MC3T3-E1 preosteoblast cell line showed good cell adhesion, proliferation, alkaline phosphatase activity and bone mineralization on the developed scaffolds.

  15. Metal levels in fodder, milk, dairy products, and tissues sampled in ovine farms of Southern Italy.

    PubMed

    Caggiano, Rosa; Sabia, Serena; D'Emilio, Mariagrazia; Macchiato, Maria; Anastasio, Aniello; Ragosta, Maria; Paino, Salvatore

    2005-09-01

    We measured Cd, Cr, Hg, Mn, and Pb levels in samples of fodder, milk, dairy products, and tissues collected from 12 ovine farms in the regions of Campania and Calabria (Southern Italy). The areas in which the farms are located show different levels of anthropogenic pressure. The main purpose of this study is the identification and the analysis of relationships among metal concentrations observed in samples representative of different links in the food chain. Particularly, we apply univariate, bivariate, and multivariate statistical techniques to identify the correlation structure of our data set and to evaluate the influence of anthropogenic activity. We discuss the results, focusing the analysis on the spatial and the temporal patterns of metal concentrations.

  16. Assessment of bioburden on human and animal tissues: part 2--results of testing of human tissue and qualification of a composite sample for routine bioburden determination.

    PubMed

    Kowalski, John B; Merritt, Karen; Gocke, David; Osborne, Joel

    2012-08-01

    A quantitative method was developed and validated to assess bioburden on tissue from human donors and to compare bioburden determination results to swab culture results from the same donor. An initial study with allograft tissue from 101 donors showed a wide range of bioburden levels; values from no colony-forming units (CFU) detected to >28,000 CFU were observed. Tissues from donors that had swab cultures negative for objectionable microorganisms generally had lower bioburden than tissues from donors where objectionable microorganisms were recovered by swab culturing. In a follow-up study with 1,445 donors, a wide range of bioburden levels was again observed on tissues from donors that were swab culture negative for objectionable microorganisms. Tissues from 885 (61%) of these donors had no recoverable bioburden (<2 CFU). Importantly, tissues from 560 (39%) of the donors had recoverable bioburden which ranged from 1 to >24,000 CFU. Identification of bioburden isolates showed a diversity of genera and species. In compliance with the recent revision of the American Association of Tissue Banks K2.210 Standard, the quantitative bioburden determination method was validated with a composite tissue sample that contains bone and soft tissue sections tested together in one extraction vessel. A recovery efficiency of 68% was validated and the composite sample was shown to be representative of all of the tissues recovered from a donor. The use of the composite sample in conjunction with the quantitative bioburden determination method will facilitate an accurate assessment of the numbers and types of contaminating microorganisms on allografts prior to disinfection/sterilization. This information will ensure that disinfection/sterilization processes are properly validated and the capability of the overall allograft process is understood on a donor by donor basis.

  17. Evaluating real-time immunohistochemistry on multiple tissue samples, multiple targets and multiple antibody labeling methods

    PubMed Central

    2013-01-01

    Background Immunohistochemistry (IHC) is a well-established method for the analysis of protein expression in tissue specimens and constitutes one of the most common methods performed in pathology laboratories worldwide. However, IHC is a multi-layered method based on subjective estimations and differences in staining and interpretation has been observed between facilities, suggesting that the analysis of proteins on tissue would benefit from protocol optimization and standardization. Here we describe how the emerging and operator independent tool of real-time immunohistochemistry (RT-IHC) reveals a time resolved description of antibody interacting with target protein in formalin fixed paraffin embedded tissue. The aim was to understand the technical aspects of RT-IHC, regarding generalization of the concept and to what extent it can be considered a quantitative method. Results Three different antibodies labeled with fluorescent or radioactive labels were applied on nine different tissue samples from either human or mouse, and the results for all RT-IHC analyses distinctly show that the method is generally applicable. The collected binding curves showed that the majority of the antibody-antigen interactions did not reach equilibrium within 3 hours, suggesting that standardized protocols for immunohistochemistry are sometimes inadequately optimized. The impact of tissue size and thickness as well as the position of the section on the glass petri dish was assessed in order for practical details to be further elucidated for this emerging technique. Size and location was found to affect signal magnitude to a larger extent than thickness, but the signal from all measurements were still sufficient to trace the curvature. The curvature, representing the kinetics of the interaction, was independent of thickness, size and position and may be a promising parameter for the evaluation of e.g. biopsy sections of different sizes. Conclusions It was found that RT-IHC can be used

  18. Feasibility of direct digital sampling for diffuse optical frequency domain spectroscopy in tissue

    NASA Astrophysics Data System (ADS)

    Roblyer, Darren; O'Sullivan, Thomas D.; Warren, Robert V.; Tromberg, Bruce J.

    2013-04-01

    Frequency domain optical spectroscopy in the diffusive regime is currently being investigated for biomedical applications including tumor detection, therapy monitoring, exercise metabolism and others. Analog homodyne or heterodyne detection of sinusoidally modulated signals has been the predominant method for measuring phase and amplitude of photon density waves that have traversed through tissue. Here we demonstrate the feasibility of utilizing direct digital sampling of modulated signals using a 3.6 gigasample/second 12 bit analog to digital converter. Digitally synthesized modulated signals between 50 MHz and 400 MHz were measured on tissue-simulating phantoms at six near-infrared wavelengths. An amplitude and phase precision of 1% and 0.6° were achieved during drift tests. Amplitude, phase, scattering and absorption values were compared with a well-characterized network analyzer-based diffuse optical device. Optical properties measured with both systems were within 3.6% for absorption and 2.8% for scattering over a range of biologically relevant values. Direct digital sampling represents a viable method for frequency domain diffuse optical spectroscopy and has the potential to reduce system complexity, size and cost.

  19. A percutaneous needle biopsy technique for sampling the supraclavicular brown adipose tissue depot of humans

    PubMed Central

    Annamalai, Palam; Chondronikola, Maria; Chao, Tony; Porter, Craig; Saraf, Manish K.; Cesani, Fernardo; Sidossis, Labros S.

    2015-01-01

    Brown adipose tissue (BAT) has been proposed as a potential target tissue against obesity and its related metabolic complications. Although the molecular and functional characteristics of BAT have been intensively studied in rodents, only a small number of studies have used human BAT specimens due to the difficulty of sampling human BAT deposits. We established a novel positron emission tomography and computed tomography-guided Bergström needle biopsy technique to acquire human BAT specimens from the supraclavicular area in human subjects. Forty-three biopsies were performed on 23 participants. The procedure was tolerated well by the majority of participants. No major complications were noted. Numbness (9.6%) and hematoma (2.3%) were the two minor complications noted, which fully resolved. Thus, the proposed biopsy technique can be considered safe with only minimal risk of adverse events. Adoption of the proposed method is expected to increase the sampling of the supraclavicular BAT depot for research purposes so as to augment the scientific knowledge of the biology of human BAT. PMID:25920777

  20. Feasibility of Direct Digital Sampling for Diffuse Optical Frequency Domain Spectroscopy in Tissue.

    PubMed

    Roblyer, Darren; O'Sullivan, Thomas D; Warren, Robert V; Tromberg, Bruce

    2013-04-01

    Frequency domain optical spectroscopy in the diffusive regime is currently being investigated for biomedical applications including tumor detection, therapy monitoring, exercise metabolism, and others. Analog homodyne or heterodyne detection of sinusoidally modulated signals have been the predominant method for measuring phase and amplitude of photon density waves that have traversed through tissue. Here we demonstrate the feasibility of utilizing direct digital sampling of modulated signals using a 3.6 Gigasample/second 12 bit Analog to Digital Converter. Digitally synthesized modulated signals between 50MHz and 400MHz were measured on tissue simulating phantoms at six near-infrared wavelengths. An amplitude and phase precision of 1% and 0.6 degrees were achieved during drift tests. Amplitude, phase, scattering and absorption values were compared with a well-characterized network analyzer based diffuse optical device. Measured optical properties measured with both systems were within 3.6% for absorption and 2.8% for scattering over a range of biologically relevant values. Direct digital sampling represents a viable method for frequency domain diffuse optical spectroscopy and has the potential to reduce system complexity, size, and cost.

  1. Additive effects of microRNAs and transcription factors on CCL2 production in human white adipose tissue.

    PubMed

    Kulyté, Agné; Belarbi, Yasmina; Lorente-Cebrián, Silvia; Bambace, Clara; Arner, Erik; Daub, Carsten O; Hedén, Per; Rydén, Mikael; Mejhert, Niklas; Arner, Peter

    2014-04-01

    Adipose tissue inflammation is present in insulin-resistant conditions. We recently proposed a network of microRNAs (miRNAs) and transcription factors (TFs) regulating the production of the proinflammatory chemokine (C-C motif) ligand-2 (CCL2) in adipose tissue. We presently extended and further validated this network and investigated if the circuits controlling CCL2 can interact in human adipocytes and macrophages. The updated subnetwork predicted that miR-126/-193b/-92a control CCL2 production by several TFs, including v-ets erythroblastosis virus E26 oncogene homolog 1 (avian) (ETS1), MYC-associated factor X (MAX), and specificity protein 12 (SP1). This was confirmed in human adipocytes by the observation that gene silencing of ETS1, MAX, or SP1 attenuated CCL2 production. Combined gene silencing of ETS1 and MAX resulted in an additive reduction in CCL2 production. Moreover, overexpression of miR-126/-193b/-92a in different pairwise combinations reduced CCL2 secretion more efficiently than either miRNA alone. However, although effects on CCL2 secretion by co-overexpression of miR-92a/-193b and miR-92a/-126 were additive in adipocytes, the combination of miR-126/-193b was primarily additive in macrophages. Signals for miR-92a and -193b converged on the nuclear factor-κB pathway. In conclusion, TF and miRNA-mediated regulation of CCL2 production is additive and partly relayed by cell-specific networks in human adipose tissue that may be important for the development of insulin resistance/type 2 diabetes.

  2. Liquid Microjunction Surface Sampling Probe Electrospray Mass Spectrometry for Detection of Drugs and Metabolites in Thin Tissue Sections

    SciTech Connect

    Van Berkel, Gary J; Kertesz, Vilmos; Koeplinger, Kenneth A.; Vavek, Marissa; Kong, Ah-Ng Tony

    2008-01-01

    A self-aspirating, liquid micro-junction surface sampling probe/electrospray emitter mass spectrometry system was demonstrated for use in the direct analysis of spotted and dosed drugs and their metabolites in thin tissue sections. Proof-of-principle sampling and analysis directly from tissue without the need for sample preparation was demonstrated first by raster scanning a region on a section of rat liver onto which reserpine was spotted. The mass spectral signal from selected reaction monitoring was used to develop a chemical image of the spotted drug on the tissue. The probe was also used to selectively spot sample areas of sagittal whole mouse body tissue sections that had been dosed orally (90 mg/kg) with R,S-sulforaphane 3 hrs prior to sacrifice. Sulforaphane and its glutathione and N-acetyl cysteine conjugates were monitored with selected reaction monitoring and detected in the stomach and various other tissues from the dosed mouse. No signal for these species was observed in the tissue from a control mouse. The same dosed tissue section was used to illustrate the possibility of obtaining a line scan across the whole body section. In total these results illustrate the potential for rapid screening of the distribution of drugs and metabolites in tissue sections with the micro-liquid junction surface sampling probe/electrospray mass spectrometry approach.

  3. Mapping international practice patterns in EUS-guided tissue sampling: outcome of a global survey

    PubMed Central

    van Riet, Priscilla A.; Cahen, Djuna L.; Poley, Jan-Werner; Bruno, Marco J.

    2016-01-01

    Background and study aims: Although Endoscopic Ultrasound (EUS)-guided tissue sampling is widely used, the optimal sampling strategy remains subject of debate. We evaluated practice patterns within the international endosonographic community. Patients and methods: An online questionnaire was sent to 400 endosonographers from the United States, Europe, and Asia. Results: A total of 186 (47 %) endosonographers participated: United States 54 (29 %), Europe 85 (46 %), and Asia 47 (25 %). European (75 %) and Asian (84 %) respondents routinely check coagulation status, whereas US respondents only check on indication (64 %, P = 0.007). While propofol sedation is standard in the United States (83 %), conscious sedation is still widely used in Europe (52 %) and Asia (84 %, P < 0.001). Overall, the 22-gauge needle is most commonly used (52 %). For fine-needle aspiration (FNA) of solid pancreatic lesions, 22-gauge (45 %) and 25-gauge (49 %) needles are used equally. For fine-needle biopsy (FNB) of solid masses, the 25-gauge device is less favored than the 22-gauge FNA device (49 % versus 21 %). The 19-gauge needle is generally used for FNB of submucosal masses (62 %). Rapid on-site pathological evaluation (ROSE) is utilized more often by US (98 %) than by European and Asian respondents (51 %, P < 0.001). Cytolyt (52 %), formalin (15 %) and alcohol (15 %) are used for FNA specimen preservation in the United States and Europe, while saline (27 %) and alcohol (38 %) are widely used in Asia (P < 0.001). Conclusions: EUS-guided tissue sampling practices vary substantially within the international endosonographic community and differ considerably from recommendations expressed in guidelines. Because the clinical relevance of these variations is largely unknown, the outcome of this survey suggests a need for further studies. PMID:27227103

  4. Concordance in diabetic foot ulceration: a cross-sectional study of agreement between wound swabbing and tissue sampling in infected ulcers.

    PubMed Central

    Nelson, E Andrea; Wright-Hughes, Alexandra; Brown, Sarah; Lipsky, Benjamin A; Backhouse, Michael; Bhogal, Moninder; Ndosi, Mwidimi; Reynolds, Catherine; Sykes, Gill; Dowson, Christopher; Edmonds, Michael; Vowden, Peter; Jude, Edward B; Dickie, Tom; Nixon, Jane

    2016-01-01

    change in therapy for 73.3% of patients (considering swab and tissue results separately), but significantly more tissue than swab samples required a change in therapy. Compared with traditional culture, the PCR technique reported additional pathogens for both swab and tissue samples in six (50%) patients and reported the same pathogens in four (33.3%) patients and different pathogens in two (16.7%) patients. The estimated healing rate was 44.5% (95% CI 38.9% to 50.1%). At 12 months post sampling, 45 (15.1%) patients had died, 52 (17.4%) patients had a lower-extremity ipsilateral amputation and 18 (6.0%) patients had revascularisation surgery. LIMITATIONS We did not investigate the potential impact of microbiological information on care. We cannot determine if the improved information yield from tissue sampling is attributable to sample collection, sample handling, processing or reporting. CONCLUSIONS Tissue sampling reported both more pathogens and more organisms overall than swabbing. Both techniques missed some organisms, with tissue sampling missing fewer than swabbing. Results from tissue sampling more frequently led to a (virtual) recommended change in therapy. Long-term prognosis for patients with an infected foot ulcer was poor. FUTURE WORK Research is needed to determine the effect of sampling/processing techniques on clinical outcomes and antibiotic stewardship. FUNDING The National Institute for Health Research Health Technology Assessment programme. PMID:27827300

  5. [Analysis of lead in unknown samples based on the standard addition method using laser induced breakdown spectroscopy].

    PubMed

    Fang, Li; Zhao, Nan-jing; Meng, De-shuo; Yuan, Jing; Tang, Jie; Wang, Yin; Yu, Yang; Ma, Ming-jun; Hu, Li; Zhang, Da-hai; Xiao, Xue; Wang, Yu; Liu, Jian-guo; Liu, Wen-qing

    2015-01-01

    The standard addition method with laser induced breakdown spectroscopy was used to analyze an unknown sample taken from a lead battery factory. the matrix influence on the results was effectively avoided when the external or internal standard method was used, and the pretreatment of samples was simple and quick. The Nd ' YAG pulse laser with wavelength 1 064 nm was used as the excitation source. The echelle spectroscopy with high resolution and wide spectral range was used as the spectral separation device, and the intensified charge coupled device (ICCD) as the spectral detection device in the experiment. The characteristic line at 405. 78 nrn was chosen as the analysis line to measure Pb concentration. Fe I : 404. 58 line was chosen as the internal standard. Pre-experiment was carried out to confirm the appropriate condition. Under the laser energy of 128. 5 mJ, the delay time of 2. 5 tps, and the gate width of 3 ps, it was determined that with the addition of Pb to the sample in the range of 0 and 25 000 mg . kg-1, there wasn't self-absorption. There was a good linear relationship between the intensity of the spectral line of 405. 78 nm and the addition of Pb. The appropriate concentration of Pb added into the sample for analysis was determined by this series of samples. On this basis, four samples were prepared with three parallel samples for each sample in order to verify the repeatability and reliability of the method, i. e. 5 000, 10 000, 15 000, 20 000 mg . kg-1 Pb was added into the original sample. The results were compared with the result of ICP-MS. The twelve samples' relative errors were between -24. 6% and 17. 6%. The average result was 43 069 mg . kg-1 with the relative error -2. 44%.

  6. Multimodal Raman-fluorescence spectroscopy of formalin fixed samples is able to discriminate brain tumors from dysplastic tissue

    NASA Astrophysics Data System (ADS)

    Anand, Suresh; Cicchi, Riccardo; Giordano, Flavio; Buccoliero, Anna Maria; Pavone, Francesco Saverio

    2014-05-01

    In the recent years, there has been a considerable surge in the application of spectroscopy for disease diagnosis. Raman and fluorescence spectra provide characteristic spectral profile related to biochemical and morphological changes when tissues progress from normal state towards malignancy. Spectroscopic techniques offer the advantage of being minimally invasive compared to traditional histopathology, real time and quantitative. In biomedical optical diagnostics, freshly excised specimens are preferred for making ex-vivo spectroscopic measurements. With regard to fresh tissues, if the lab is located far away from the clinic it could pose a problem as spectral measurements have to be performed immediately after dissection. Tissue samples are usually placed in a fixative agent such as 4% formaldehyde to preserve the samples before processing them for routine histopathological studies. Fixation prevents the tissues from decomposition by arresting autolysis. In the present study, we intend to investigate the possibility of using formalin fixed samples for discrimination of brain tumours from dysplastic tissue using Raman and fluorescence spectroscopy. Formalin fixed samples were washed with phosphate buffered saline for about 5 minutes in order to remove the effects of formalin during spectroscopic measurements. In case of fluorescence spectroscopy, changes in spectral profile have been observed in the region between 550-670 nm between dysplastic and tumor samples. For Raman measurements, we found significant differences in the spectral profiles between dysplasia and tumor. In conclusion, formalin fixed samples can be potentially used for the spectroscopic discrimination of tumor against dysplastic tissue in brain samples.

  7. Macro-to-micro interfacing to microfluidic channels using 3D-printed templates: application to time-resolved secretion sampling of endocrine tissue.

    PubMed

    Brooks, Jessica C; Ford, Katarena I; Holder, Dylan H; Holtan, Mark D; Easley, Christopher J

    2016-10-21

    Employing 3D-printed templates for macro-to-micro interfacing, a passively operated polydimethysiloxane (PDMS) microfluidic device was designed for time-resolved secretion sampling from primary murine islets and epidiymal white adipose tissue explants. Interfacing in similar devices is typically accomplished through manually punched or drilled fluidic reservoirs. We previously introduced the concept of using hand fabricated polymer inserts to template cell culture and sampling reservoirs into PDMS devices, allowing rapid stimulation and sampling of endocrine tissue. However, fabrication of the fluidic reservoirs was time consuming, tedious, and was prone to errors during device curing. Here, we have implemented computer-aided design and 3D printing to circumvent these fabrication obstacles. In addition to rapid prototyping and design iteration advantages, the ability to match these 3D-printed interface templates with channel patterns is highly beneficial. By digitizing the template fabrication process, more robust components can be produced with reduced fabrication variability. Herein, 3D-printed templates were used for sculpting millimetre-scale reservoirs into the above-channel, bulk PDMS in passively-operated, eight-channel devices designed for time-resolved secretion sampling of murine tissue. Devices were proven functional by temporally assaying glucose-stimulated insulin secretion from <10 pancreatic islets and glycerol secretion from 2 mm adipose tissue explants, suggesting that 3D-printed interface templates could be applicable to a variety of cells and tissue types. More generally, this work validates desktop 3D printers as versatile interfacing tools in microfluidic laboratories.

  8. Attitudes towards transfers of human tissue samples across borders: An international survey of researchers and policy makers in five countries

    PubMed Central

    2010-01-01

    Background Sharing of tissue samples for research and disease surveillance purposes has become increasingly important. While it is clear that this is an area of intense, international controversy, there is an absence of data about what researchers themselves and those involved in the transfer of samples think about these issues, particularly in developing countries. Methods A survey was carried out in a number of Asian countries and in Egypt to explore what researchers and others involved in research, storage and transfer of human tissue samples thought about some of the issues related to sharing of such samples. Results The results demonstrated broad agreement with the positions taken by developing countries in the current debate, favoring quite severe restrictions on the use of samples by developed countries. Conclusions It is recommended that an international agreement is developed on what conditions should be attached to any sharing of human tissue samples across borders. PMID:20843366

  9. One-step labeling of degenerative neurons in unfixed brain tissue samples using Fluoro-Jade C.

    PubMed

    Gu, Qiang; Schmued, Larry C; Sarkar, Sumit; Paule, Merle G; Raymick, Bryan

    2012-06-30

    Neurodegeneration is the underlying cause of a vast majority of neurological disorders and often a result of brain trauma, stroke, or neurotoxic insult. Here we describe a simple method for labeling degenerating neurons in unfixed brain tissue samples. This method could provide a new avenue for identifying and harvesting degenerative neurons from unfixed brain tissues for subsequent molecular analyses.

  10. Alaska Marine Mammal Tissue Archival Project: Sample inventory and results of analyses of selected samples for organic compounds and trace elements

    SciTech Connect

    Becker, P.R.; Wise, S.A.; Schantz, M.M.; Koster, B.J.; Zeisler, R.

    1992-02-01

    In 1987, the Alaska Marine Mammal Tissue Archival Project (AMMTAP) was established as part of the National Biomonitoring Specimen Bank (NBSB) program at the National Institute of Standards and Technology (NIST).The purpose of the AMMTAP was to establish a representative collection of Alaska marine mammal tissues for future contaminant analyses and documentation of long-term trends in environmental quality. Since 1987, specimens have been collected from 65 animals (seven species) from six different sites. The report contains the current sample inventory and the results of the analysis of selected samples for the measurement of inorganic and organic compounds.

  11. Preservation of nucleic acids and tissue morphology in paraffin-embedded clinical samples: comparison of five molecular fixatives.

    PubMed

    Staff, Synnöve; Kujala, Paula; Karhu, Ritva; Rökman, Annika; Ilvesaro, Joanna; Kares, Saara; Isola, Jorma

    2013-09-01

    Formalin fixation preserves tissue morphology at the expense of macromolecule integrity. Freshly frozen samples are the golden standard for DNA and RNA analyses but require laborious deep-freezing and frozen sectioning for morphological studies. Alternative tissue stabilisation methods are therefore needed. We analysed the preservation of nucleic acids, immunohistochemical staining properties and tissue morphology in paraffin-embedded clinical tissue samples fixed with Z7, RCL2, PAXgene, Allprotect and RNAlater. Formalin-fixed and deep-frozen samples were used as controls. Immunohistochemical analyses showed good preservation of antigenicity in all except Allprotect and RNAlater-fixed samples. RNA quality, based on RNA integrity number value by Bioanalyzer, was comparable with freshly frozen samples only in PAXgene-fixed samples. According to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses, RNA from PAXgene samples yielded results similar to freshly frozen samples. No difference between fixatives was seen in DNA analyses (PCR and real-time PCR). In conclusion, PAXgene seems to be superior to other molecular fixatives and formaldehyde.

  12. Design and implementation of coded aperture coherent scatter spectral imaging of cancerous and healthy breast tissue samples

    PubMed Central

    Lakshmanan, Manu N.; Greenberg, Joel A.; Samei, Ehsan; Kapadia, Anuj J.

    2016-01-01

    Abstract. A scatter imaging technique for the differentiation of cancerous and healthy breast tissue in a heterogeneous sample is introduced in this work. Such a technique has potential utility in intraoperative margin assessment during lumpectomy procedures. In this work, we investigate the feasibility of the imaging method for tumor classification using Monte Carlo simulations and physical experiments. The coded aperture coherent scatter spectral imaging technique was used to reconstruct three-dimensional (3-D) images of breast tissue samples acquired through a single-position snapshot acquisition, without rotation as is required in coherent scatter computed tomography. We perform a quantitative assessment of the accuracy of the cancerous voxel classification using Monte Carlo simulations of the imaging system; describe our experimental implementation of coded aperture scatter imaging; show the reconstructed images of the breast tissue samples; and present segmentations of the 3-D images in order to identify the cancerous and healthy tissue in the samples. From the Monte Carlo simulations, we find that coded aperture scatter imaging is able to reconstruct images of the samples and identify the distribution of cancerous and healthy tissues (i.e., fibroglandular, adipose, or a mix of the two) inside them with a cancerous voxel identification sensitivity, specificity, and accuracy of 92.4%, 91.9%, and 92.0%, respectively. From the experimental results, we find that the technique is able to identify cancerous and healthy tissue samples and reconstruct differential coherent scatter cross sections that are highly correlated with those measured by other groups using x-ray diffraction. Coded aperture scatter imaging has the potential to provide scatter images that automatically differentiate cancerous and healthy tissue inside samples within a time on the order of a minute per slice. PMID:26962543

  13. Design and implementation of coded aperture coherent scatter spectral imaging of cancerous and healthy breast tissue samples.

    PubMed

    Lakshmanan, Manu N; Greenberg, Joel A; Samei, Ehsan; Kapadia, Anuj J

    2016-01-01

    A scatter imaging technique for the differentiation of cancerous and healthy breast tissue in a heterogeneous sample is introduced in this work. Such a technique has potential utility in intraoperative margin assessment during lumpectomy procedures. In this work, we investigate the feasibility of the imaging method for tumor classification using Monte Carlo simulations and physical experiments. The coded aperture coherent scatter spectral imaging technique was used to reconstruct three-dimensional (3-D) images of breast tissue samples acquired through a single-position snapshot acquisition, without rotation as is required in coherent scatter computed tomography. We perform a quantitative assessment of the accuracy of the cancerous voxel classification using Monte Carlo simulations of the imaging system; describe our experimental implementation of coded aperture scatter imaging; show the reconstructed images of the breast tissue samples; and present segmentations of the 3-D images in order to identify the cancerous and healthy tissue in the samples. From the Monte Carlo simulations, we find that coded aperture scatter imaging is able to reconstruct images of the samples and identify the distribution of cancerous and healthy tissues (i.e., fibroglandular, adipose, or a mix of the two) inside them with a cancerous voxel identification sensitivity, specificity, and accuracy of 92.4%, 91.9%, and 92.0%, respectively. From the experimental results, we find that the technique is able to identify cancerous and healthy tissue samples and reconstruct differential coherent scatter cross sections that are highly correlated with those measured by other groups using x-ray diffraction. Coded aperture scatter imaging has the potential to provide scatter images that automatically differentiate cancerous and healthy tissue inside samples within a time on the order of a minute per slice.

  14. Sample data processing in an additive and reproducible taxonomic workflow by using character data persistently linked to preserved individual specimens

    PubMed Central

    Kilian, Norbert; Henning, Tilo; Plitzner, Patrick; Müller, Andreas; Güntsch, Anton; Stöver, Ben C.; Müller, Kai F.; Berendsohn, Walter G.; Borsch, Thomas

    2015-01-01

    We present the model and implementation of a workflow that blazes a trail in systematic biology for the re-usability of character data (data on any kind of characters of pheno- and genotypes of organisms) and their additivity from specimen to taxon level. We take into account that any taxon characterization is based on a limited set of sampled individuals and characters, and that consequently any new individual and any new character may affect the recognition of biological entities and/or the subsequent delimitation and characterization of a taxon. Taxon concepts thus frequently change during the knowledge generation process in systematic biology. Structured character data are therefore not only needed for the knowledge generation process but also for easily adapting characterizations of taxa. We aim to facilitate the construction and reproducibility of taxon characterizations from structured character data of changing sample sets by establishing a stable and unambiguous association between each sampled individual and the data processed from it. Our workflow implementation uses the European Distributed Institute of Taxonomy Platform, a comprehensive taxonomic data management and publication environment to: (i) establish a reproducible connection between sampled individuals and all samples derived from them; (ii) stably link sample-based character data with the metadata of the respective samples; (iii) record and store structured specimen-based character data in formats allowing data exchange; (iv) reversibly assign sample metadata and character datasets to taxa in an editable classification and display them and (v) organize data exchange via standard exchange formats and enable the link between the character datasets and samples in research collections, ensuring high visibility and instant re-usability of the data. The workflow implemented will contribute to organizing the interface between phylogenetic analysis and revisionary taxonomic or monographic work

  15. Micro-scaled high-throughput digestion of plant tissue samples for multi-elemental analysis

    PubMed Central

    Hansen, Thomas H; Laursen, Kristian H; Persson, Daniel P; Pedas, Pai; Husted, Søren; Schjoerring, Jan K

    2009-01-01

    Background Quantitative multi-elemental analysis by inductively coupled plasma (ICP) spectrometry depends on a complete digestion of solid samples. However, fast and thorough sample digestion is a challenging analytical task which constitutes a bottleneck in modern multi-elemental analysis. Additional obstacles may be that sample quantities are limited and elemental concentrations low. In such cases, digestion in small volumes with minimum dilution and contamination is required in order to obtain high accuracy data. Results We have developed a micro-scaled microwave digestion procedure and optimized it for accurate elemental profiling of plant materials (1-20 mg dry weight). A commercially available 64-position rotor with 5 ml disposable glass vials, originally designed for microwave-based parallel organic synthesis, was used as a platform for the digestion. The novel micro-scaled method was successfully validated by the use of various certified reference materials (CRM) with matrices rich in starch, lipid or protein. When the micro-scaled digestion procedure was applied on single rice grains or small batches of Arabidopsis seeds (1 mg, corresponding to approximately 50 seeds), the obtained elemental profiles closely matched those obtained by conventional analysis using digestion in large volume vessels. Accumulated elemental contents derived from separate analyses of rice grain fractions (aleurone, embryo and endosperm) closely matched the total content obtained by analysis of the whole rice grain. Conclusion A high-throughput micro-scaled method has been developed which enables digestion of small quantities of plant samples for subsequent elemental profiling by ICP-spectrometry. The method constitutes a valuable tool for screening of mutants and transformants. In addition, the method facilitates studies of the distribution of essential trace elements between and within plant organs which is relevant for, e.g., breeding programmes aiming at improvement of the

  16. Integrated genome-wide analysis of genomic changes and gene regulation in human adrenocortical tissue samples.

    PubMed

    Gara, Sudheer Kumar; Wang, Yonghong; Patel, Dhaval; Liu-Chittenden, Yi; Jain, Meenu; Boufraqech, Myriem; Zhang, Lisa; Meltzer, Paul S; Kebebew, Electron

    2015-10-30

    To gain insight into the pathogenesis of adrenocortical carcinoma (ACC) and whether there is progression from normal-to-adenoma-to-carcinoma, we performed genome-wide gene expression, gene methylation, microRNA expression and comparative genomic hybridization (CGH) analysis in human adrenocortical tissue (normal, adrenocortical adenomas and ACC) samples. A pairwise comparison of normal, adrenocortical adenomas and ACC gene expression profiles with more than four-fold expression differences and an adjusted P-value < 0.05 revealed no major differences in normal versus adrenocortical adenoma whereas there are 808 and 1085, respectively, dysregulated genes between ACC versus adrenocortical adenoma and ACC versus normal. The majority of the dysregulated genes in ACC were downregulated. By integrating the CGH, gene methylation and expression profiles of potential miRNAs with the gene expression of dysregulated genes, we found that there are higher alterations in ACC versus normal compared to ACC versus adrenocortical adenoma. Importantly, we identified several novel molecular pathways that are associated with dysregulated genes and further experimentally validated that oncostatin m signaling induces caspase 3 dependent apoptosis and suppresses cell proliferation. Finally, we propose that there is higher number of genomic changes from normal-to-adenoma-to-carcinoma and identified oncostatin m signaling as a plausible druggable pathway for therapeutics.

  17. Stable isotope analysis of 1987-1991 zooplankton samples and bowhead whale tissues. Final report

    SciTech Connect

    Schell, D.M.

    1992-06-01

    Stable isotope analyses of bowhead whale tissue samples and bowhead whale prey organisms collected over the years 1987 to 1991 were used to provide detail on the isotope ratio gradients evident in the arctic Alaskan zooplankton and to verify previous findings regarding the growth rates and age determination techniques developed for bowhead whales. Zooplankton of the Bering and Chukchi seas are enriched in (13)C relative to the eastern Beaufort Sea. The analysis of baleen from bowhead whales taken between 1987 to 1990 indicate that the whales are slow-growing and the young animals between year one and about six to seven years of age, undergo a period of little or no linear growth. The authors estimate that bowheads require 16-18 years to reach the length of sexual maturity, i.e., 13-14 m. From baleen Delta(13C) cycles, a 20 year record of the isotope ratios in the phytoplankton of the northern Bering and Chukchi seas was constructed. The long-term record has been compared with the temperature anomalies in surface waters of the Bering Sea. The Delta(13C) of the zooplankton is inversely correlated with temperature and refutes current models attempting to relate ocean temperature, and atmospheric carbon dioxide levels with the Delta(13C) of ocean sediment organic matter.

  18. Gene expression profiling of human breast tissue samples using SAGE-Seq.

    PubMed

    Wu, Zhenhua Jeremy; Meyer, Clifford A; Choudhury, Sibgat; Shipitsin, Michail; Maruyama, Reo; Bessarabova, Marina; Nikolskaya, Tatiana; Sukumar, Saraswati; Schwartzman, Armin; Liu, Jun S; Polyak, Kornelia; Liu, X Shirley

    2010-12-01

    We present a powerful application of ultra high-throughput sequencing, SAGE-Seq, for the accurate quantification of normal and neoplastic mammary epithelial cell transcriptomes. We develop data analysis pipelines that allow the mapping of sense and antisense strands of mitochondrial and RefSeq genes, the normalization between libraries, and the identification of differentially expressed genes. We find that the diversity of cancer transcriptomes is significantly higher than that of normal cells. Our analysis indicates that transcript discovery plateaus at 10 million reads/sample, and suggests a minimum desired sequencing depth around five million reads. Comparison of SAGE-Seq and traditional SAGE on normal and cancerous breast tissues reveals higher sensitivity of SAGE-Seq to detect less-abundant genes, including those encoding for known breast cancer-related transcription factors and G protein-coupled receptors (GPCRs). SAGE-Seq is able to identify genes and pathways abnormally activated in breast cancer that traditional SAGE failed to call. SAGE-Seq is a powerful method for the identification of biomarkers and therapeutic targets in human disease.

  19. Effects of addition of tissue-type plasminogen activator in in vitro fertilization medium on bovine embryo development and quality.

    PubMed

    Krania, F; Dovolou, E; Rekkas, C A; Theodosiadou, E K; Pappas, I; Amiridis, G S

    2015-02-01

    Plasminogen activators/Plasmin system plays pivotal role in regulating reproductive functions of mammals. Here, we examined the effects of modification of in vitro fertilization medium (IVF medium) with the addition of tissue-type plasminogen activator (t-PA), on bovine embryo development and quality, assessed by quantification of expression of various genes related to metabolism, oxidation, implantation and apoptosis. In addition, plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were measured in the spent media. After conventional IVM, 2016 cumulus-oocyte complexes (COCs) were divided into four groups with modified composition of the IVF medium containing t-PA and/or its inhibitor epsilon-aminocaproic acid (control, t-PA, t-PA+ε-ACA, ε-ACA). Presumptive zygotes were cultured for 8 days in synthetic oviductal fluid (SOF) medium; gene expression studies were carried out on morulae and blastocysts. t-PA alone significantly suppressed cleavage and blastocyst formation rates, but this effect was neutralized by the addition of ε-ACA. PAA in the treated group was significantly reduced by ε-ACA, but without total elimination. Significant differences were detected in the expression of genes related to apoptosis and/or cell cycle arrest (BAX, BCL2L1, KAT2B) between embryos produced in t-PA-modified media and controls, giving an overall notion that the inferior developmental competence of treated embryos may be attributed to apoptotic phenomena induced by t-PA. In conclusion, it appears that excessive t-PA content in the IVF media, suppresses blastocyst formation rate, possibly due to induction of apoptotic phenomena.

  20. Comparison between whole mount tissue preparations and virtual tissue microarray samples for measuring Ki-67 and apoptosis indices in human bladder cancer

    PubMed Central

    Oshiro, Hisashi; Czerniak, Bogdan A.; Sakamaki, Kentaro; Tsuta, Koji; Bondaruk, Jolanta; Keyhani, Afsaneh; Dinney, Colin P.; Nagai, Takeshi; Kamat, Ashish M.

    2016-01-01

    Abstract Recent tissue microarray (TMA)-based studies have shown that cell proliferation- and apoptosis-related biomarkers are associated with clinical outcomes in patients with bladder urothelial carcinoma. However, little is known about the differences in these biomarker measurements between whole mount tissue preparations and TMAs. This study aimed to elucidate the discrepancy in the measurements of Ki-67 indices (KIs) and apoptosis indices (AIs) between whole mount tissue preparations and TMAs of bladder urothelial carcinoma samples. Whole mount tissue preparations for Ki-67 immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling were made from 30 patients who underwent transurethral resection of bladder urothelial carcinoma. Digital microscopy-assisted virtual TMAs, consisting of 3 small round areas (1 or 0.6 mm in diameter), were generated from the same whole mount tissue preparations. The measurement results in highly reactive areas of biomarkers were compared between the whole mount tissue preparation- and the TMA-based methods. Bland–Altman plot analysis, regression analysis, and Kendall τ were performed to investigate differences in the measurement results, systematic biases, and correlations between biomarkers. Although the Bland–Altman plot analysis demonstrated that almost all the plots were within the limits of agreement, fixed biases were detected in the 1- and 0.6-mm TMAs for the KI (0.181 and 0.222, respectively) and the AI (0.055 and 0.063, respectively). Proportional biases were also detected in the 1- and 0.6-mm TMAs for the AI (P < 0.001 and P < 0.001, respectively). Furthermore, positive correlations between KIs and AIs were observed in whole mount tissue preparations (r = 0.260, P = 0.044) and in the 1 mm TMAs (r = 0.375, P = 0.004); however, no such correlation was observed in the 0.6 mm TMAs. Our study suggests that the measurement results for certain biomarkers of bladder

  1. Comparison between whole mount tissue preparations and virtual tissue microarray samples for measuring Ki-67 and apoptosis indices in human bladder cancer: A cross-sectional study.

    PubMed

    Oshiro, Hisashi; Czerniak, Bogdan A; Sakamaki, Kentaro; Tsuta, Koji; Bondaruk, Jolanta; Keyhani, Afsaneh; Dinney, Colin P; Nagai, Takeshi; Kamat, Ashish M

    2016-08-01

    Recent tissue microarray (TMA)-based studies have shown that cell proliferation- and apoptosis-related biomarkers are associated with clinical outcomes in patients with bladder urothelial carcinoma. However, little is known about the differences in these biomarker measurements between whole mount tissue preparations and TMAs. This study aimed to elucidate the discrepancy in the measurements of Ki-67 indices (KIs) and apoptosis indices (AIs) between whole mount tissue preparations and TMAs of bladder urothelial carcinoma samples.Whole mount tissue preparations for Ki-67 immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling were made from 30 patients who underwent transurethral resection of bladder urothelial carcinoma. Digital microscopy-assisted virtual TMAs, consisting of 3 small round areas (1 or 0.6 mm in diameter), were generated from the same whole mount tissue preparations. The measurement results in highly reactive areas of biomarkers were compared between the whole mount tissue preparation- and the TMA-based methods. Bland-Altman plot analysis, regression analysis, and Kendall τ were performed to investigate differences in the measurement results, systematic biases, and correlations between biomarkers.Although the Bland-Altman plot analysis demonstrated that almost all the plots were within the limits of agreement, fixed biases were detected in the 1- and 0.6-mm TMAs for the KI (0.181 and 0.222, respectively) and the AI (0.055 and 0.063, respectively). Proportional biases were also detected in the 1- and 0.6-mm TMAs for the AI (P < 0.001 and P < 0.001, respectively). Furthermore, positive correlations between KIs and AIs were observed in whole mount tissue preparations (r = 0.260, P = 0.044) and in the 1 mm TMAs (r = 0.375, P = 0.004); however, no such correlation was observed in the 0.6 mm TMAs.Our study suggests that the measurement results for certain biomarkers of bladder urothelial

  2. Epigenome-wide profiling of DNA methylation in paired samples of adipose tissue and blood

    PubMed Central

    Huang, Yen-Tsung; Chu, Su; Loucks, Eric B.; Lin, Chien-Ling; Eaton, Charles B.; Buka, Stephen L.; Kelsey, Karl T.

    2016-01-01

    ABSTRACT Many epigenetic association studies have attempted to identify DNA methylation markers in blood that are able to mirror those in target tissues. Although some have suggested potential utility of surrogate epigenetic markers in blood, few studies have collected data to directly compare DNA methylation across tissues from the same individuals. Here, epigenomic data were collected from adipose tissue and blood in 143 subjects using Illumina HumanMethylation450 BeadChip array. The top axis of epigenome-wide variation differentiates adipose tissue from blood, which is confirmed internally using cross-validation and externally with independent data from the two tissues. We identified 1,285 discordant genes and 1,961 concordant genes between blood and adipose tissue. RNA expression data of the two classes of genes show consistent patterns with those observed in DNA methylation. The discordant genes are enriched in biological functions related to immune response, leukocyte activation or differentiation, and blood coagulation. We distinguish the CpG-specific correlation from the within-subject correlation and emphasize that the magnitude of within-subject correlation does not guarantee the utility of surrogate epigenetic markers. The study reinforces the critical role of DNA methylation in regulating gene expression and cellular phenotypes across tissues, and highlights the caveats of using methylation markers in blood to mirror the corresponding profile in the target tissue. PMID:26891033

  3. Epigenome-wide profiling of DNA methylation in paired samples of adipose tissue and blood.

    PubMed

    Huang, Yen-Tsung; Chu, Su; Loucks, Eric B; Lin, Chien-Ling; Eaton, Charles B; Buka, Stephen L; Kelsey, Karl T

    2016-03-03

    Many epigenetic association studies have attempted to identify DNA methylation markers in blood that are able to mirror those in target tissues. Although some have suggested potential utility of surrogate epigenetic markers in blood, few studies have collected data to directly compare DNA methylation across tissues from the same individuals. Here, epigenomic data were collected from adipose tissue and blood in 143 subjects using Illumina HumanMethylation450 BeadChip array. The top axis of epigenome-wide variation differentiates adipose tissue from blood, which is confirmed internally using cross-validation and externally with independent data from the two tissues. We identified 1,285 discordant genes and 1,961 concordant genes between blood and adipose tissue. RNA expression data of the two classes of genes show consistent patterns with those observed in DNA methylation. The discordant genes are enriched in biological functions related to immune response, leukocyte activation or differentiation, and blood coagulation. We distinguish the CpG-specific correlation from the within-subject correlation and emphasize that the magnitude of within-subject correlation does not guarantee the utility of surrogate epigenetic markers. The study reinforces the critical role of DNA methylation in regulating gene expression and cellular phenotypes across tissues, and highlights the caveats of using methylation markers in blood to mirror the corresponding profile in the target tissue.

  4. Optical coherence tomography detection of shear wave propagation in inhomogeneous tissue equivalent phantoms and ex-vivo carotid artery samples.

    PubMed

    Razani, Marjan; Luk, Timothy W H; Mariampillai, Adrian; Siegler, Peter; Kiehl, Tim-Rasmus; Kolios, Michael C; Yang, Victor X D

    2014-03-01

    In this work, we explored the potential of measuring shear wave propagation using optical coherence elastography (OCE) in an inhomogeneous phantom and carotid artery samples based on a swept-source optical coherence tomography (OCT) system. Shear waves were generated using a piezoelectric transducer transmitting sine-wave bursts of 400 μs duration, applying acoustic radiation force (ARF) to inhomogeneous phantoms and carotid artery samples, synchronized with a swept-source OCT (SS-OCT) imaging system. The phantoms were composed of gelatin and titanium dioxide whereas the carotid artery samples were embedded in gel. Differential OCT phase maps, measured with and without the ARF, detected the microscopic displacement generated by shear wave propagation in these phantoms and samples of different stiffness. We present the technique for calculating tissue mechanical properties by propagating shear waves in inhomogeneous tissue equivalent phantoms and carotid artery samples using the ARF of an ultrasound transducer, and measuring the shear wave speed and its associated properties in the different layers with OCT phase maps. This method lays the foundation for future in-vitro and in-vivo studies of mechanical property measurements of biological tissues such as vascular tissues, where normal and pathological structures may exhibit significant contrast in the shear modulus.

  5. Rapid sample preparation for determination of iron in tissue by closed-vessel digestion and microwave energy.

    PubMed

    Van Wyck, D B; Schifman, R B; Stivelman, J C; Ruiz, J; Martin, D

    1988-06-01

    We developed a rapid acid-digestion method for preparing tissue samples for iron determination. Specimens were digested in nitric acid and hydrogen peroxide under high temperature and pressure in closed Teflon vessels, with microwave energy. Analysis for iron in 25- to 250-mg portions of digested bovine liver powder (National Bureau of Standards Certified Reference Material no. 1577a) showed excellent linearity ([predicted] = 1.007[actual] - 0.166 micrograms per sample) and analytical recovery (98%). Precision (CV) was 5.4% when iron content was 10 micrograms per sample. Assaying split samples of mouse tissues, we found a close correlation between iron concentrations obtained with closed vs open vessels ([closed] = 0.878[open] + 68 micrograms/g, r = 0.994, range 400-4600 micrograms/g dry weight). In contrast to time-consuming conventional procedures for tissue dissolution, closed-vessel digestion with microwave energy dramatically shortens time for tissue preparation, minimizes use of caustic acid, reduces risk of sample loss or contamination, and yields accurate and reproducible results.

  6. Genomic DNA isolation of Acrocomia aculeata (Arecaceae) from leaf and stipe tissue samples for PCR analysis.

    PubMed

    Lanes, E C M; Nick, C; Kuki, K N; Freitas, R D; Motoike, S Y

    2013-09-23

    Macaw palm, Acrocomia aculeata is an oleaginous species of the Arecaceae family; it has been identified as one of the most promising plants for sustainable production of renewable energy, especially biodiesel. We developed an efficient protocol of genomic DNA extraction for A. aculeata using leaf and stipe tissues, based on the cationic hexadecyltrimethylammonium bromide method, and we evaluated the quantity, purity, and integrity of the resultant DNA. We also determined whether these procedures interfere with PCR amplification using SSR molecular markers. The lowest concentration of DNA was obtained from stipe tissues (135 ng/μL), while fresh leaf tissues provided the highest concentration of DNA (650 ng/μL). Good quality DNA was obtained from fresh leaf, lyophilized leaf, and stipe tissues (relative purity, 1.79-1.89 nm). Differences in quantity and quality of DNA extracted from different tissues did not interfere with general patterns of PCR amplification based on SSR markers.

  7. Additional variability at the D12S391 STR locus in an Austrian population sample: sequencing data and allele distribution.

    PubMed

    Glock, B; Dauber, E M; Schwartz, D W; Mayr, W R

    1997-12-01

    The highly polymorphic STR locus D12S391 was investigated in an Austrian population sample (N = 150) by PCR-amplification, comparative detection on native and denaturing polyacrylamide gels and solid phase single stranded sequencing of three size variant alleles and several additional alleles. A total of 15 alleles, distinguishable by size under denaturing conditions, could be detected. No deviations from Hardy-Weinberg equilibrium were observed in the population investigated (P = 0.52). Sequencing of size variants designated 17.3 and 18.3 showed an incomplete (GAT) repeat unit at position two of the tandem region. Additional new sequence variants due to varying compositions of the number of (AGAT) and (AGAC) repeats could be identified. Due to distinct electrophoretical mobilities of alleles of the same size but different sequence structures, denaturing detection conditions should be employed when the aim is standardization.

  8. Virus isolation vs RT-PCR: which method is more successful in detecting VHSV and IHNV in fish tissue sampled under field conditions?

    PubMed

    Knüsel, R; Bergmann, S M; Einer-Jensen, K; Casey, J; Segner, H; Wahli, T

    2007-09-01

    This study compared the results of reverse transcription-polymerase chain reaction (RT-PCR) and traditional virus isolation on cell culture in detection of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV). RT-PCR was used for 172 tissue sample pools (total of 859 fish) originating from a field survey on the occurrence of VHSV and IHNV in farmed and wild salmonids in Switzerland. These samples represented all sites with fish that were either identified as virus-positive by means of virus isolation (three sites, four positive tissue sample pools) and/or demonstrated positive anti-VHSV-antibody titres (83 sites, 121 positive blood samples) in a serum plaque neutralization test (SPNT). The RT-PCR technique confirmed the four VHSV-positive tissue sample pools detected by virus isolation and additionally identified one VHSV-positive sample that showed positive anti-VHSV-AB titres, but was negative in virus isolation. With IHNV, RT-PCR detected two positive samples not identified by virus isolation while in these fish the SPNT result had been questionable. One of the IHNV-positive samples represents the first detection of IHNV-RNA in wild brown trout in Switzerland. Compared to SPNT, the RT-PCR method detected, as with virus isolation, a much lower number of positive cases; reasons for this discrepancy are discussed. Our results indicate that RT-PCR can not only be successfully applied in field surveys, but may also be slightly more sensitive than virus isolation. However, in a titration experiment under laboratory conditions, the sensitivity of RT-PCR was not significantly higher when compared with virus isolation.

  9. Suitability of faeces and tissue samples as a basis for non-invasive sampling for African swine fever in wild boar.

    PubMed

    de Carvalho Ferreira, H C; Weesendorp, E; Quak, S; Stegeman, J A; Loeffen, W L A

    2014-08-27

    A challenging aspect of ASFV control in wild boar populations is the design and implementation of effective surveillance and monitoring programmes, both for early warning, and to determine the ongoing epidemiological situation in an infected population. Testing blood samples requires invasive sampling strategies like hunting or capture of wild boar. Besides being biased towards healthy animals, such strategies are also linked to further spread of the virus. Non-invasive sampling strategies would increase the reliability of surveillance of ASFV in wild boar populations, without the negative side effects. This study evaluates the potential of faeces and tissue samples as a basis for non-invasive sampling strategies for ASFV in wild boar. In the acute phase (0-21 days after infection), in comparison with virus detection in blood, virus can be detected in faeces 50-80% of the time. This percentage decreases to below 10% for the subacute/chronic phase. ASFV DNA is quite stable in faeces. Half-lives range from more than 2 years at temperature up to 12°C, to roughly 15 days at temperatures of 30°C. In tissue samples, stored at 20°C, half-lives mostly range from 1.7 to 7.4 days. The sample of preference is the spleen, where the highest titres and highest half-life of ASFV DNA are observed. The level and duration of excretion of ASFV in the faeces, combined with the stability of the DNA, suggest that sampling of faeces could be the basis for a non-invasive sampling strategy to monitor ASFV in wild boar.

  10. Detection and assay of cis- and trans-isomers of 4-methylaminorex in urine, plasma and tissue samples.

    PubMed

    Kankaanpää, A; Meririnne, E; Ellermaa, S; Ariniemi, K; Seppälä, T

    2001-09-15

    The 4-methylaminorex (4-MAX) is an amphetamine-related psychostimulant drug that has appeared on the clandestine market with a street name of "U4Euh". This compound exists as four stereoisomers, trans-4R,5R, trans-4S,5S, cis-4R,5S and cis-4S,5R, of which the cis forms have been classified as Schedule I substances in the US. The increasing variety of designer drugs has highlighted the importance of detection, identification, and quantitative measurement of these drugs, including 4-MAX, in biological samples. In the present study, the isomers of 4-MAX were detected in urine of rats treated with the drugs by some but not all of the on-site immunoassays tested, mainly as amphetamine or methamphetamine. To facilitate identification of 4-MAX by laboratories specialized in drug analysis, the electron-ionization mass spectrum and TLC data for underivatized 4-MAX using a routine laboratory drug-screening procedure is provided. In addition, a GC/MS method is described for the quantitative determination of cis- and trans-4-MAX as tert-butyldimethylsilyl-derivatives in plasma, urine and tissue.

  11. Development of laboratory control samples for the ICP-ES determination of nutrient elements in rat tissues

    NASA Astrophysics Data System (ADS)

    Wolnik, Karen A.; Rader, Jeanne I.; Gaston, Cynthia M.; Fricke, Fred L.

    Laboratory control samples have been prepared from rabbit bones and duodenum and from bovine kidney and spleen for use in quality control and development of methodology for the inductively coupled plasma emission spectroscopy (ICP-ES) determination of 9 elements in weanling rat tissues. Analysis by ICP-ES following wet acid digestion was used to characterize these control samples with respect to mineral content and homogeneity. Results of the investigation of alternative pretreatment techniques are included.

  12. Right ventricular systolic pressure measurements in combination with harvest of lung and immune tissue samples in mice.

    PubMed

    Chen, Wen-Chi; Park, Sung-Hyun; Hoffman, Carol; Philip, Cecil; Robinson, Linda; West, James; Grunig, Gabriele

    2013-01-16

    The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes. Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system. Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues. The

  13. A total extract dot blot hybridization procedure for mRNA quantitation in small samples of tissues or cultured cells.

    PubMed

    Grimes, A; McArdle, H J; Mercer, J F

    1988-08-01

    A simple method for the estimation of specific mRNA concentrations in small tissue samples (as little as 1 mg) or cultured cells (lower limit 10(5) cells) is described. Guanidine hydrochloride extracts of whole cells or tissues are applied directly onto nitrocellulose and hybridized with the appropriate nick-translated probe. Loading according to DNA content allows expression of the result as concentration per cell. Hybridizing with a ribosomal RNA probe allows expression of results relative to rRNA and estimation of the RNA/DNA ratio in the sample. We describe the application of this procedure to the measurement of ceruloplasmin mRNA in tissues and cultured hepatocytes.

  14. Single-pixel hyperspectral imaging for real-time cancer detection: detecting damage in ex vivo porcine tissue samples

    NASA Astrophysics Data System (ADS)

    Peller, Joseph; Farahi, Faramarz; Trammell, Susan R.

    2016-03-01

    We are developing a single-pixel hyperspectral imaging system based on compressive sensing that acquires spatial and spectral information simultaneously. Our spectral imaging system uses autofluorescencent emission from collagen (400 nm) and NAD(P)H (475 nm), as well as, differences in the optical reflectance spectra as diagnostics for differentiating between healthy and diseased tissue. In this study, we demonstrate the ability of our imaging system to discriminate between healthy and damaged porcine epidermal tissue. Healthy porcine epidermal tissue samples (n=11) were imaged ex vivo using our hyperspectral system. The amount of NAD(P)H emission and the reflectance properties were approximately constant across the surface of healthy tissue samples. The tissue samples were then thermally damaged using an 1850 nm thulium fiber laser and re-imaged after laser irradiation. The damaged regions were clearly visible in the hyperspectral images as the thermal damage altered the fluorescent emission of NAD(P)H and changed the scattering properties of the tissue. The extent of the damaged regions was determined based on the hyperspectral images and these estimates were compared to damage extents measured in white light images acquired with a traditional camera. The extent of damage determined via hyperspectral imaging was in good agreement with estimates based on white light imaging indicating that our system is capable of differentiating between healthy and damaged tissue. Possible applications of our single pixel hyperspectral imaging system range from real-time determination of tumor margins during surgery to the use of this technique in the pathology lab to aid with cancer diagnosis and staging.

  15. Development and application of specific cytokine assays in tissue samples from a bottlenose dolphin with hyperinsulinemia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic inflammation has been associated with insulin resistance and type 2 diabetes in humans. Postmortem hepatic and splenic tissue from a 46-year old geriatric male bottlenose dolphin (Tursiops truncatus) with insulin resistance (chronic hyperinsulinemia with hyperglycemia) , chronic = inflamma...

  16. The bio-gripper: a fluid-driven micro-manipulator of living tissue constructs for additive bio-manufacturing.

    PubMed

    Ip, Blanche C; Cui, Francis; Tripathi, Anubhav; Morgan, Jeffrey R

    2016-05-25

    We previously developed the Bio-Pick, Place, and Perfuse (Bio-P3) instrument to fabricate large perfusable tissue constructs by stacking and aligning scaffold-free living microtissues with integrated lumens. The Bio-P3 required an actuating mechanism to manipulate living microtissues of various sizes and shapes that are fragile, and must remain in an aqueous environment. The optical transparency of the Bio-P3 gripping device was essential to provide unobstructed visuals for accurate alignment of microtissues. We previously engineered a pilot fluid force-driven bio-gripper that can pick-and-place microtissue in planar position without causing cellular damage by pulling culture medium through track-etched membrane-integrated cell culture inserts. In this study, we invented a new flexible bio-gripper design that maximized the bio-gripper utilities. We utilized experimental approaches, multivariate analyzes, and theoretical modeling to elucidate how membrane characteristics (pore size, pore density, membrane thickness, membrane area, and surface chemistry) altered bio-gripper robustness and the flow rate (Q(c)) required for successful gripping. We devised two standardized tests and synthetic parts that mimicked microtissues, to systematically quantify bio-gripper performance. All thirteen syringe pump-driven bio-grippers except one successfully gripped and released synthetic parts with values of Q(c) that coincided with our mathematical simulation of the fluid mechanics of gripping. The bio-gripper could grip synthetic parts of various sizes, shapes and masses, demonstrating the robustness of the actuating mechanism. Multivariate analysis of experimental data indicated that both membrane porosity and thickness modulated Q(c), and in addition, revealed that membrane pore density determined membrane optical transparency. Fabricating large tissue constructs requires repeated stacking of microtissues. We showed that one bio-gripper could pick-and-place living microtissues

  17. Measurement of characteristic prompt gamma rays emitted from oxygen and carbon in tissue-equivalent samples during proton beam irradiation.

    PubMed

    Polf, Jerimy C; Panthi, Rajesh; Mackin, Dennis S; McCleskey, Matt; Saastamoinen, Antti; Roeder, Brian T; Beddar, Sam

    2013-09-07

    The purpose of this work was to characterize how prompt gamma (PG) emission from tissue changes as a function of carbon and oxygen concentration, and to assess the feasibility of determining elemental concentration in tissues irradiated with proton beams. For this study, four tissue-equivalent water-sucrose samples with differing densities and concentrations of carbon, hydrogen, and oxygen were irradiated with a 48 MeV proton pencil beam. The PG spectrum emitted from each sample was measured using a high-purity germanium detector, and the absolute detection efficiency of the detector, average beam current, and delivered dose distribution were also measured. Changes to the total PG emission from (12)C (4.44 MeV) and (16)O (6.13 MeV) per incident proton and per Gray of absorbed dose were characterized as a function of carbon and oxygen concentration in the sample. The intensity of the 4.44 MeV PG emission per incident proton was found to be nearly constant for all samples regardless of their carbon concentration. However, we found that the 6.13 MeV PG emission increased linearly with the total amount (in grams) of oxygen irradiated in the sample. From the measured PG data, we determined that 1.64 × 10(7) oxygen PGs were emitted per gram of oxygen irradiated per Gray of absorbed dose delivered with a 48 MeV proton beam. These results indicate that the 6.13 MeV PG emission from (16)O is proportional to the concentration of oxygen in tissue irradiated with proton beams, showing that it is possible to determine the concentration of oxygen within tissues irradiated with proton beams by measuring (16)O PG emission.

  18. Measurement of characteristic prompt gamma rays emitted from oxygen and carbon in tissue-equivalent samples during proton beam irradiation

    PubMed Central

    Polf, Jerimy C; Panthi, Rajesh; Mackin, Dennis S; McCleskey, Matt; Saastamoinen, Antti; Roeder, Brian T; Beddar, Sam

    2013-01-01

    The purpose of this work was to characterize how prompt gamma (PG) emission from tissue changes as a function of carbon and oxygen concentration, and to assess the feasibility of determining elemental concentration in tissues irradiated with proton beams. For this study, four tissue-equivalent water-sucrose samples with differing densities and concentrations of carbon, hydrogen, and oxygen were irradiated with a 48 MeV proton pencil beam. The PG spectrum emitted from each sample was measured using a high-purity germanium detector, and the absolute detection efficiency of the detector, average beam current, and delivered dose distribution were also measured. Changes to the total PG emission from 12C (4.44 MeV) and 16O (6.13 MeV) per incident proton and per Gray of absorbed dose were characterized as a function of carbon and oxygen concentration in the sample. The intensity of the 4.44 MeV PG emission per incident proton was found to be nearly constant for all samples regardless of their carbon concentration. However, we found that the 6.13 MeV PG emission increased linearly with the total amount (in grams) of oxygen irradiated in the sample. From the measured PG data, we determined that 1.64 × 107 oxygen PGs were emitted per gram of oxygen irradiated per Gray of absorbed dose delivered with a 48 MeV proton beam. These results indicate that the 6.13 MeV PG emission from 16O is proportional to the concentration of oxygen in tissue irradiated with proton beams, showing that it is possible to determine the concentration of oxygen within tissues irradiated with proton beams by measuring 16O PG emission. PMID:23920051

  19. Simultaneous determination of perfluorinated compounds and their potential precursors in mussel tissue and fish muscle tissue and liver samples by liquid chromatography-electrospray-tandem mass spectrometry.

    PubMed

    Zabaleta, I; Bizkarguenaga, E; Prieto, A; Ortiz-Zarragoitia, M; Fernández, L A; Zuloaga, O

    2015-03-27

    An analytical method for the simultaneous determination in fish liver and muscle tissue and mussel samples of 14 perfluorinated compounds (PFCs), including three perfluoroalkylsulfonates (PFSAs), seven perfluorocarboxylic acids (PFCAs), three perfluorophosphonic acids (PFPAs) and perfluorooctanesulfonamide (PFOSA), and 10 potential precursors, including four polyfluoroalkyl phosphates (PAPs), four fluorotelomer saturated acids (FTCAs) and two fluorotelomer unsaturated acids (FTUCAs), was developed in the present work. Different clean-up strategies by means of solid-phase extraction (SPE) using a mix-mode weak anion exchanger (WAX), reverse phase Envi-Carb or a combination of them was optimized and evaluated for the clean-up of focused ultrasonic solid-liquid (FUSLE) extracts before the analysis by liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS/MS). Mix-mode WAX coupled in-line to Envi-Carb was finally selected since it rendered the cleanest extracts and minimum matrix effect. The FUSLE-SPE-LC-MS/MS methodology was validated in terms of recovery, precision and method detection limits (MDLs). Apparent recovery values in the 65-116%, 59-119% and 67-126% range and MDLs in the 0.1-2.7 ng/g, 0.1-3.8 ng/g and 0.2-3.1ng/g range were obtained for liver, mussel and fish muscle tissue samples, respectively. The method developed was applied to the analysis of grey mullet liver (Chelon labrosus) and mussel (Mytilus galloprovincialis) samples from the Basque Coast (North of Spain) and Yellowfin tuna muscle tissue (Thunnus albacares) samples from the Indian Ocean. To the best of our knowledge this is the first method that describes the simultaneous determination of 14 PFCs and 10 potential precursors in fish liver, fish muscle tissue and mussel samples. Besides, this is the first time that 8:2 monosubstituted polyfluorodecyl phosphate (8:2 monoPAP) and 8:2 disubstituted polyfluorodecyl phosphate (8:2 diPAP) were detected in mussel and tuna samples

  20. Detection of bovine viral diarrhoea virus infected cattle--testing tissue samples derived from ear tagging using an Erns capture ELISA.

    PubMed

    Kuhne, S; Schroeder, C; Holmquist, G; Wolf, G; Horner, S; Brem, G; Ballagi, A

    2005-08-01

    A new diagnostic approach testing tissue samples derived from cattle ear tagging for bovine viral diarrhoea virus (BVDV) antigen in a commercially available antigen capture enzyme-linked immunosorbent assay (ACE) was developed. To validate this method, 99 positive and 469 negative samples were tested. With those samples the assay yielded a sensitivity of 100% and specificity of >or=99.6%. Serum and ear tissue samples from 11 persistently infected (PI) BVDV calves were tested. While serum samples were negative after intake of colostrum, the ear tissue samples could be detected positive for BVDV all the time. Testing multiple samples derived from the same ear from PI cattle yielded positive results and low variation. Using cattle ear tags combining the ear tag application with sampling of a small ear tissue plug and testing those tissue samples with an ACE could be a reliable and economic way of BVDV testing.

  1. A novel device to stretch multiple tissue samples with variable patterns: application for mRNA regulation in tissue-engineered constructs.

    PubMed

    Imsirovic, Jasmin; Derricks, Kelsey; Buczek-Thomas, Jo Ann; Rich, Celeste B; Nugent, Matthew A; Suki, Béla

    2013-01-01

    A broad range of cells are subjected to irregular time varying mechanical stimuli within the body, particularly in the respiratory and circulatory systems. Mechanical stretch is an important factor in determining cell function; however, the effects of variable stretch remain unexplored. In order to investigate the effects of variable stretch, we designed, built and tested a uniaxial stretching device that can stretch three-dimensional tissue constructs while varying the strain amplitude from cycle to cycle. The device is the first to apply variable stretching signals to cells in tissues or three dimensional tissue constructs. Following device validation, we applied 20% uniaxial strain to Gelfoam samples seeded with neonatal rat lung fibroblasts with different levels of variability (0%, 25%, 50% and 75%). RT-PCR was then performed to measure the effects of variable stretch on key molecules involved in cell-matrix interactions including: collagen 1α, lysyl oxidase, α-actin, β1 integrin, β3 integrin, syndecan-4, and vascular endothelial growth factor-A. Adding variability to the stretching signal upregulated, downregulated or had no effect on mRNA production depending on the molecule and the amount of variability. In particular, syndecan-4 showed a statistically significant peak at 25% variability, suggesting that an optimal variability of strain may exist for production of this molecule. We conclude that cycle-by-cycle variability in strain influences the expression of molecules related to cell-matrix interactions and hence may be used to selectively tune the composition of tissue constructs.

  2. Comparing citrated native, kaolin-activated, and tissue factor-activated samples and determining intraindividual variability for feline thromboelastography.

    PubMed

    Banerjee, Amrita; Blois, Shauna L; Wood, R Darren

    2011-11-01

    Thromboelastography (TEG) is a point-of-care whole blood test of hemostasis. While TEG is becoming more widely used in veterinary medicine, few studies describe the use of TEG in cats. The objectives of the current study were to: 1) document the range of TEG variables produced in healthy cats using 3 sample types (citrated native, kaolin-activated, and tissue factor-activated), and 2) determine if there was a significant difference between 2 separate samples obtained from individual healthy cats on the same day. Jugular venipuncture was performed in 20 cats, and citrated blood collected for TEG. TEG analysis was performed on citrated native, kaolin-activated, and tissue factor-activated blood for each sample. Two hours later, the procedure was repeated from the opposite jugular vein, yielding a total of 120 analyses. Reaction time (R), alpha angle (α), kappa value (κ), and maximum amplitude (MA) were recorded from each tracing. No significant differences were found between TEG tracings from the first and second venipuncture samples. Significant differences were found between sample types for R, α, κ, and MA. Means for citrated native/kaolin-activated/tissue factor-activated methods were R = 4.1/3.7/0.6 min; κ = 2.5/1.8/2.2 min; α = 59.9/65.1/70.4 degrees; MA = 47.4/49.9/44.7 mm. A limitation of this study was the small number of cats used. Thromboelastography analysis may be a suitable method of evaluating hemostasis in cats.

  3. Probing focal cortical dysplasia in formalin fixed samples using tissue optical spectroscopy

    NASA Astrophysics Data System (ADS)

    Anand, Suresh; Cicchi, Riccardo; Giordano, Flavio; Buccoliero, Anna Maria; Conti, Valerio; Guerrini, Renzo; Pavone, Francesco Saverio

    2016-03-01

    Focal cortical dysplasia (FCD) is one of most common causes of intractable epilepsy in pediatric population and these are often insensitive to anti-epileptic drugs. FCD is characterized by a disarray in localized regions of the cerebral cortex and abnormal neurons which results them to misfire with incorrect signals. Resective neurosurgery to remove or disconnect the affected parts from the rest of the brain seems to be a viable option to treat FCD. Before neurosurgery the subject could undergo imaging studies including magnetic resonance imaging (MRI) or computed tomography (CT) scans. On the downside FCD could be elusive in MRI images and may be practically invisible in CT scans. Furthermore, unnecessary removal of normal tissues is to be taken into consideration as this could lead to neurological defects. In this context, optical spectroscopy have been widely investigated as an alternative technique for the detection of abnormal tissues in different organ sites. Disease progression is accompanied by a number of architectural, biochemical and morphological changes. These variations are reflected in the spectral intensity and line shape. Here, in this proof of concept study we propose to investigate the application of tissue optical spectroscopy based on fluorescence excitation at two wavelength 378 and 445 nm coupled along with Raman spectroscopy for the detection of FCD on formalin fixed tissue specimens from pediatric subjects. For fluorescence at both the excitation wavelengths FCD showed a decreased intensity at longer wavelength when compared to normal tissues. Also, differences exist in the Raman spectral profiles of normal and FCD.

  4. Optical parameter measurement of highly diffusive tissue body phantoms with specifically designed sample holder for photo diagnostic and PDT applications

    NASA Astrophysics Data System (ADS)

    Rehman, A.; Rehman, K.; Anwar, S.; Firdous, S.; Nawaz, M.

    2015-12-01

    Knowledge of optical properties (absorption coefficients, scattering Coefficients, and anisotropy) is necessary for understanding light tissue interactions. Optical parameters define the behavior of light in the tissues. Intralipid and Indian ink are well-established tissue body phantoms. Quantitative characterization of biological tissues in terms of optical properties is achieved with integrating sphere. However, samples having significantly higher scattering and absorption coefficients such as malignant tissues potentially reduce the signal to noise ratio (SNR) and accuracy of integrating sphere. We have measured the diffuse reflection and transmission of these phantoms by placing them in integrating sphere at 632.8 nm and then applied IAD method to determine the optical properties tissue phantoms composed of Indian ink (1.0%) and Intralipid (20%). We have fabricated a special sample holder with thin microscopic cover slips which can be used to measure signal from highly concentrated intralipid and Indian ink solutions. Experiments conducted with various phantoms reveal significant improvement of SNR for a wide range of optical properties. This approach opens up a field for potential applications in measurement of optical properties of highly diffusive biological tissues. For 20% intralipid μa =0.112+/-0.046 cm-1 and μs =392.299+/-10.090 cm-1 at 632.8 nm and for 1.0% Indian ink μa =9.808+/-0.490 cm-1 and μs =1.258+/-0.063 cm-1 at same wavelength. System shows good repeatability and reproducibility within 4.9% error. Work may have important biomedical applications in photo-diagnosis and Photodynamic therapy.

  5. Translational intracerebral hemorrhage: a need for transparent descriptions of fresh tissue sampling and preclinical model quality.

    PubMed

    Chang, Che-Feng; Cai, Li; Wang, Jian

    2015-10-01

    For years, strategies have been proposed to improve translational success in stroke research by improving the quality of animal studies. However, articles that report preclinical intracerebral hemorrhage (ICH) studies continue to lack adequate qualitative and quantitative descriptions of fresh brain tissue collection. They also tend to lack transparency about animal model quality. We conducted a systematic review of 82 ICH research articles to determine the level of detail reported for brain tissue collection. We found that only 24 (29 %) reported the volume, weight, or thickness of tissue collected and a specific description of the anatomical location. Thus, up to 71 % of preclinical ICH research articles did not properly define how fresh specimens were collected for biochemical measurements. Such omissions may impede reproducibility of results between laboratories. Although existing criteria have improved the quality of preclinical stroke studies, ICH researchers need to identify specific guidelines and strategies to avoid pitfalls, minimize bias, and increase reproducibility in this field.

  6. Translational Intracerebral Hemorrhage: A Need for Transparent Descriptions of Fresh Tissue Sampling and Preclinical Model Quality

    PubMed Central

    Chang, Che-Feng; Cai, Li; Wang, Jian

    2015-01-01

    For years, strategies have been proposed to improve translational success in stroke research by improving the quality of animal studies. However, articles that report preclinical intracerebral hemorrhage (ICH) studies continue to lack adequate qualitative and quantitative descriptions of fresh brain tissue collection. They also tend to lack transparency about animal model quality. We conducted a systematic review of 82 ICH research articles to determine the level of detail reported for brain tissue collection. We found that only 24 (29%) reported the volume, weight, or thickness of tissue collected and a specific description of the anatomical location. Thus, up to 71% of preclinical ICH research articles did not properly define how fresh specimens were collected for biochemical measurements. Such omissions may impede reproducibility of results between laboratories. Although existing criteria have improved the quality of preclinical stroke studies, ICH researchers need to identify specific guidelines and strategies to avoid pitfalls, minimize bias, and increase reproducibility in this field. PMID:25907620

  7. Analysis of CT Numbers and Relative Proton Stopping Powers for Real Tissue Samples

    SciTech Connect

    Ryazantsev, Oleg; Karpunin, Vladimir; Haibullin, Vadim; Matusova, Tatiana

    2010-01-05

    The accuracy of computer planning of clinical proton dose distribution is partly determined by the precision of the conversion of CT Hounsfield Units to relative proton stopping powers. The calibration curves from three different sources were compared. We have found about 5% differences between proton stopping power values in the range from -700 HU to 0 HU. Calibration data for several soft tissues and phantom materials were also experimentally measured. The data for the tissues are in a good agreement with the calibration curves however data for phantom materials have significant deviations.

  8. Towards a phylogenetic generic classification of Thelypteridaceae: Additional sampling suggests alterations of neotropical taxa and further study of paleotropical genera.

    PubMed

    Almeida, Thaís Elias; Hennequin, Sabine; Schneider, Harald; Smith, Alan R; Batista, João Aguiar Nogueira; Ramalho, Aline Joseph; Proite, Karina; Salino, Alexandre

    2016-01-01

    Thelypteridaceae is one of the largest fern families, having about 950 species and a cosmopolitan distribution but with most species occurring in tropical and subtropical regions. Its generic classification remains controversial, with different authors recognizing from one up to 32 genera. Phylogenetic relationships within the family have not been exhaustively studied, but previous studies have confirmed the monophyly of the lineage. Thus far, sampling has been inadequate for establishing a robust hypothesis of infrafamilial relationships within the family. In order to understand phylogenetic relationships within Thelypteridaceae and thus to improve generic reclassification, we expand the molecular sampling, including new samples of Old World taxa and, especially, many additional neotropical representatives. We also explore the monophyly of exclusively or mostly neotropical genera Amauropelta, Goniopteris, Meniscium, and Steiropteris. Our sampling includes 68 taxa and 134 newly generated sequences from two plastid genomic regions (rps4-trnS and trnL-trnF), plus 73 rps4 and 72 trnL-trnF sequences from GenBank. These data resulted in a concatenated matrix of 1980 molecular characters for 149 taxa. The combined data set was analyzed using maximum parsimony and bayesian inference of phylogeny. Our results are consistent with the general topological structure found in previous studies, including two main lineages within the family: phegopteroid and thelypteroid. The thelypteroid lineage comprises two clades; one of these included the segregates Metathelypteris, Coryphopteris, and Amauropelta (including part of Parathelypteris), whereas the other comprises all segregates of Cyclosorus s.l., such as Goniopteris, Meniscium, and Steiropteris (including Thelypteris polypodioides, previously incertae sedis). The three mainly neotropical segregates were found to be monophyletic but nested in a broadly defined Cyclosorus. The fourth mainly neotropical segregate, Amauropelta

  9. Measurement of intravenously administered γ-Fe2O3 particle amount in mice tissues using vibrating sample magnetometer.

    PubMed

    Kishimoto, Mikio; Miyamoto, Ryoichi; Oda, Tatsuya; Ohara, Yusuke; Yanagihara, Hideto; Ohkohchi, Nobuhiro; Kita, Eiji

    2014-12-01

    Dispersions of platelet γ-Fe2O3 particles 30-50nm in size were intravenously administered to mice and the amount of particles accumulated in each tissue was obtained by magnetization measurement using a vibrating sample magnetometer. Background noise was greatly reduced by measuring dried tissues under a magnetic field of 500 Oe so that the effect of diamagnetism was slight. Remarkable particle accumulation was observed in the liver and spleen. Considerable particle accumulation was observed in the lung when a large quantity of γ-Fe2 O3 particles was administered. There was no significant particle accumulation in the kidney and heart.

  10. Comparison of Accuracy of Whole-Exome Sequencing with Formalin-Fixed Paraffin-Embedded and Fresh Frozen Tissue Samples

    PubMed Central

    Kwon, Mi Jeong; Kim, Ryong Nam; Kim, Yu Jin; Song, Ji-Young; Jung, Kyung Soo; Shin, Young Kee

    2015-01-01

    Formalin fixing with paraffin embedding (FFPE) has been a standard sample preparation method for decades, and archival FFPE samples are still very useful resources. Nonetheless, the use of FFPE samples in cancer genome analysis using next-generation sequencing, which is a powerful technique for the identification of genomic alterations at the nucleotide level, has been challenging due to poor DNA quality and artificial sequence alterations. In this study, we performed whole-exome sequencing of matched frozen samples and FFPE samples of tissues from 4 cancer patients and compared the next-generation sequencing data obtained from these samples. The major differences between data obtained from the 2 types of sample were the shorter insert size and artificial base alterations in the FFPE samples. A high proportion of short inserts in the FFPE samples resulted in overlapping paired reads, which could lead to overestimation of certain variants; >20% of the inserts in the FFPE samples were double sequenced. A large number of soft clipped reads was found in the sequencing data of the FFPE samples, and about 30% of total bases were soft clipped. The artificial base alterations, C>T and G>A, were observed in FFPE samples only, and the alteration rate ranged from 200 to 1,200 per 1M bases when sequencing errors were removed. Although high-confidence mutation calls in the FFPE samples were compatible to that in the frozen samples, caution should be exercised in terms of the artifacts, especially for low-confidence calls. Despite the clearly observed artifacts, archival FFPE samples can be a good resource for discovery or validation of biomarkers in cancer research based on whole-exome sequencing. PMID:26641479

  11. Evaluation of a novel tagging and tissue preservation system for potential use in forensic sample collection.

    PubMed

    Grassberger, Martin; Stein, Christina; Hanslik, Stefan; Hochmeister, Manfred

    2005-07-16

    The authors describe a new, easy-to-use barcode-based tissue collection, preservation and body tracking system, which might prove instrumental in the containment of mass fatalities such as aircraft accidents, war related accidents, environmental disasters (e.g. earthquakes, hurricanes, and floods) terrorist bombings or mass murders.

  12. Identification of immune cell infiltration in hematoxylin-eosin stained breast cancer samples: texture-based classification of tissue morphologies

    NASA Astrophysics Data System (ADS)

    Turkki, Riku; Linder, Nina; Kovanen, Panu E.; Pellinen, Teijo; Lundin, Johan

    2016-03-01

    The characteristics of immune cells in the tumor microenvironment of breast cancer capture clinically important information. Despite the heterogeneity of tumor-infiltrating immune cells, it has been shown that the degree of infiltration assessed by visual evaluation of hematoxylin-eosin (H and E) stained samples has prognostic and possibly predictive value. However, quantification of the infiltration in H and E-stained tissue samples is currently dependent on visual scoring by an expert. Computer vision enables automated characterization of the components of the tumor microenvironment, and texture-based methods have successfully been used to discriminate between different tissue morphologies and cell phenotypes. In this study, we evaluate whether local binary pattern texture features with superpixel segmentation and classification with support vector machine can be utilized to identify immune cell infiltration in H and E-stained breast cancer samples. Guided with the pan-leukocyte CD45 marker, we annotated training and test sets from 20 primary breast cancer samples. In the training set of arbitrary sized image regions (n=1,116) a 3-fold cross-validation resulted in 98% accuracy and an area under the receiver-operating characteristic curve (AUC) of 0.98 to discriminate between immune cell -rich and - poor areas. In the test set (n=204), we achieved an accuracy of 96% and AUC of 0.99 to label cropped tissue regions correctly into immune cell -rich and -poor categories. The obtained results demonstrate strong discrimination between immune cell -rich and -poor tissue morphologies. The proposed method can provide a quantitative measurement of the degree of immune cell infiltration and applied to digitally scanned H and E-stained breast cancer samples for diagnostic purposes.

  13. Bisulfite-Based DNA Methylation Analysis from Recent and Archived Formalin-Fixed, Paraffin Embedded Colorectal Tissue Samples.

    PubMed

    Kalmár, Alexandra; Péterfia, Bálint; Hollósi, Péter; Wichmann, Barnabás; Bodor, András; Patai, Árpád V; Schöller, Andrea; Krenács, Tibor; Tulassay, Zsolt; Molnár, Béla

    2015-09-01

    We aimed to test the applicability of formalin-fixed and paraffin-embedded (FFPE) tissue samples for gene specific DNA methylation analysis after using two commercially available DNA isolation kits. Genomic DNA was isolated from 5 colorectal adenocarcinomas and 5 normal adjacent tissues from "recent", collected within 6 months, and "archived", collected more than 5 years ago, FFPE tissues using either High Pure FFPET DNA Isolation kit or QIAamp DNA FFPE Tissue kit. DNA methylation analysis of MAL, SFRP1 and SFRP2 genes, known to be hypermethylated in CRC, was performed using methylation-sensitive high resolution melting (MS-HRM) analysis and sequencing. QIAamp (Q) method resulted in slightly higher recovery in archived (HP: 1.22 ± 3.18 μg DNA; Q: 3.00 ± 4.04 μg DNA) and significantly (p < 0.05) higher recovery in recent samples compared to High Pure method (HP) (HP: 4.10 ± 2.91 μg DNA; Q: 11.51 ± 7.50 μg DNA). Both OD260/280 and OD260/230 ratios were lower, but still high in the High Pure isolated archived and recent samples compared to those isolated with QIAamp. Identical DNA methylation patterns were detected for all 3 genes tested by MS-HRM with both isolation kits in the recent group. However, despite of higher DNA recovery in QIAamp slightly more reproducible methylation results were obtained from High Pure isolated archived samples. Sequencing confirmed DNA hypermethylation in CRCs. In conclusion, reproducible DNA methylation patterns were obtained from recent samples using both isolation kits. However, long term storage may affect the reliability of the results leading to moderate differences between the efficiency of isolation kits.

  14. Enrichment of single neurons and defined brain regions from human brain tissue samples for subsequent proteome analysis.

    PubMed

    Molina, Mariana; Steinbach, Simone; Park, Young Mok; Yun, Su Yeong; Di Lorenzo Alho, Ana Tereza; Heinsen, Helmut; Grinberg, Lea T; Marcus, Katrin; Leite, Renata E Paraizo; May, Caroline

    2015-07-01

    Brain function in normal aging and neurological diseases has long been a subject of interest. With current technology, it is possible to go beyond descriptive analyses to characterize brain cell populations at the molecular level. However, the brain comprises over 100 billion highly specialized cells, and it is a challenge to discriminate different cell groups for analyses. Isolating intact neurons is not feasible with traditional methods, such as tissue homogenization techniques. The advent of laser microdissection techniques promises to overcome previous limitations in the isolation of specific cells. Here, we provide a detailed protocol for isolating and analyzing neurons from postmortem human brain tissue samples. We describe a workflow for successfully freezing, sectioning and staining tissue for laser microdissection. This protocol was validated by mass spectrometric analysis. Isolated neurons can also be employed for western blotting or PCR. This protocol will enable further examinations of brain cell-specific molecular pathways and aid in elucidating distinct brain functions.

  15. Decreased Expression of Inhibitor of Caspase-Activated DNase (ICAD) in Renal Cell Carcinoma – Tissue Microarray of Human Samples

    PubMed Central

    Rajandram, Retnagowri; Razack, Azad H. A.; Ng, Keng Lim

    2016-01-01

    Although primary localised tumours of renal cell carcinoma (RCC) can be treated relatively successfully with surgery, metastatic RCC has poor prognosis because of late diagnosis and resistance to therapies. In the present study, we were interested in profiling the protein expression of “inhibitor of caspase-activated DNase” (ICAD), an apoptosis inhibitor, in kidney cancer and its paired normal kidney. Immunohistochemistry with automated batch staining and morphometry using digital pathology were used to compare ICAD in 121 RCC specimens with their paired normal kidney tissue. Tissue microarray of formalin-fixed, paraffin-embedded archival tissue was used. Intensity and localisation of ICAD were compared between normal and cancer samples, and against grading within the cancers. The results demonstrated that, in this cohort, ICAD was highly expressed in the proximal tubular epithelium of normal kidney, and significantly decreased in clear cell RCC tissue (p < 0.05) as well as other subtypes of RCC (p < 0.01) compared with normal kidney. There was a tendency towards nuclear localisation of ICAD in clear cell RCC, but not in other subtypes of RCC. No significant association was found between ICAD intensity and grade of RCC. In summary, down-regulation of ICAD occurs in RCC. ICAD normally inhibits DNA fragmentation and apoptosis; thus, its down-regulation was unexpected in a cancer known for its resistance to apoptosis. However, these RCC samples were from primary, not metastatic, RCC sites, and down-regulated ICAD may be part of a progressive pathway that promotes RCC metastasis.

  16. Evaluation of a digestion assay and determination of sample size and tissue for the reliable detection of Trichinella larvae in walrus meat.

    PubMed

    Leclair, Daniel; Forbes, Lorry B; Suppa, Sandy; Gajadhar, Alvin A

    2003-03-01

    A digestion assay was validated for the detection of Trichinella larvae in walrus (Odobenus rosmarus) meat, and appropriate samples for testing were determined using tissues from infected walruses harvested for food. Examination of muscles from 3 walruses showed that the tongue consistently contained approximately 2-6 times more larvae than the pectoral and intercostal muscles. Comparison of numbers of larvae in the root, body, and apex of the tongue from 3 walruses failed to identify a predilection site within the tongue, but the apex was considered an optimal tissue because of the high larval density within the tongue and the ease of collection. All 31 spiked samples weighing 50 g each and containing between 0.1 and 0.4 larvae per gram (lpg) were correctly identified as infected, indicating that the sensitivity of this procedure is adequate for diagnostic use. A sample size of 10 g consistently detected larvae in 2 walrus tongues containing > or = 0.3 lpg (n = 40), and until additional data are available, sample sizes from individual walrus tongues should be a minimum of 10 g. This study provides the preliminary data that were used for the development of a food safety analytical protocol for the detection of Trichinella in walrus meat in arctic communities.

  17. Micro-Raman spectroscopy of tissue samples for oral pathology follow-up monitoring

    NASA Astrophysics Data System (ADS)

    Delfino, I.; Camerlingo, C.; Zenone, F.; Perna, G.; Capozzi, V.; Cirillo, N.; Gaeta, G. M.; Lepore, M.

    2010-04-01

    An "in vitro" study of Raman spectra from oral human tissues is reported in order to the develop a diagnostic method suitable for "in vivo" oral pathology follow-up. The investigated pathology is Pemphigus Vulgaris (PV) for which new techniques for guiding and monitoring therapy would be particularly useful. Raman spectra were obtained in the wavenumber regions from 1000 to 1800 cm-1 and 2700 to 3200 cm-1 from tissues from patients at different stages of pathology (active PV, under therapy and in PV remission stage) as confirmed by histopathological and immunofluorescence analysis. Differences in the spectra depending on tissue illness stage arise in 1150-1250 cm-1 (amide III) and 1420-1450 cm-1 (CH3 deformation) regions and around 1650 cm-1 (amide I) and 2930 cm-1 (CH3 symmetric stretch). A wavelet deconvolution procedure was applied to the spectra for better discriminating among the three different stages of illness and a linear regression analysis was used to fully exploit the content of information of Raman spectra.

  18. Automated Liquid Microjunction Surface Sampling-HPLC-MS/MS Analysis of Drugs and Metabolites in Whole-Body Thin Tissue Sections

    SciTech Connect

    Kertesz, Vilmos; Van Berkel, Gary J

    2013-01-01

    A fully automated liquid extraction-based surface sampling system utilizing a commercially available autosampler coupled to high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) detection is reported. Discrete spots selected for droplet-based sampling and automated sample queue generation for both the autosampler and MS were enabled by using in-house developed software. In addition, co-registration of spatially resolved sampling position and HPLC-MS information to generate heatmaps of compounds monitored for subsequent data analysis was also available in the software. The system was evaluated with whole-body thin tissue sections from propranolol dosed rat. The hands-free operation of the system was demonstrated by creating heatmaps of the parent drug and its hydroxypropranolol glucuronide metabolites with 1 mm resolution in the areas of interest. The sample throughput was approximately 5 min/sample defined by the time needed for chromatographic separation. The spatial distributions of both the drug and its metabolites were consistent with previous studies employing other liquid extraction-based surface sampling methodologies.

  19. Facilitated Leaching of Additive-Derived PBDEs from Plastic by Seabirds' Stomach Oil and Accumulation in Tissues.

    PubMed

    Tanaka, Kosuke; Takada, Hideshige; Yamashita, Rei; Mizukawa, Kaoruko; Fukuwaka, Masa-Aki; Watanuki, Yutaka

    2015-10-06

    Our previous study suggested the transfer of polybrominated diphenyl ether (PBDE) flame retardants from ingested plastics to seabirds' tissues. To understand how the PBDEs are transferred, we studied leaching from plastics into digestive fluids. We hypothesized that stomach oil, which is present in the digestive tract of birds in the order Procellariiformes, acts as an organic solvent, facilitating the leaching of hydrophobic chemicals. Pieces of plastic compounded with deca-BDE were soaked in several leaching solutions. Trace amounts were leached into distilled water, seawater, and acidic pepsin solution. In contrast, over 20 times as much material was leached into stomach oil, and over 50 times as much into fish oil (a major component of stomach oil). Analysis of abdominal adipose, liver tissue, and ingested plastics from 18 wild seabirds collected from the North Pacific Ocean showed the occurrence of deca-BDE or hexa-BDEs in both the tissues and the ingested plastics in three of the birds, suggesting transfer from the plastic to the tissues. In birds with BDE209 in their tissues, the dominance of BDE207 over other nona-BDE isomers suggested biological debromination at the meta position. Model calculation of PBDE exposure to birds based on the results of the leaching experiments combined with field observations suggested the dominance of plastic-mediated internal exposure to BDE209 over exposure via prey.

  20. Storage and shipping of tissue samples for DNA analyses: A case study on earthworms☆

    PubMed Central

    Straube, Daniela; Juen, Anita

    2013-01-01

    Nowadays, molecular analyses play an important role in studies of soil dwelling animals, for example in taxonomy, phylogeography or food web analyses. The quality of the DNA, used for later molecular analyses, is an important factor and depends on collection and preservation of samples prior to DNA extraction. Ideally, DNA samples are frozen immediately upon collection, but if samples are collected in the field, suitable preservation methods might be limited due to unavailability of resources or remote field sites. Moreover, shipping samples over long distances can cause loss of DNA quality e.g. by thawing or leaking of preservation liquid. In this study we use earthworms, a key organism in soil research, to compare three different DNA preservation methods – freezing at −20 °C, storing in 75% ethanol, and freeze drying. Samples were shipped from the United States of America to Austria. The DNA of the samples was extracted using two different extraction methods, peqGOLD™ and Chelex® 100. The DNA amplification success was determined by amplifying four DNA fragments of different length. The PCR amplification success is significantly influenced by preservation method and extraction method and differed significantly depending on the length of the DNA fragment. Freeze drying samples was the best preservation method when samples were extracted using the silica based extraction method peqGOLD™. For samples that were extracted with Chelex® 100, storage in ethanol was the best preservation method. However, the overall amplification success was significantly lower for the extraction procedure based on Chelex® 100. The detection of the small DNA fragments was higher and independent from the extraction method, while the amplification success was significantly reduced for the longer DNA fragments. We recommend freeze drying of DNA samples, especially when they have to be shipped for longer distances. No special packaging or declaration is needed for freeze dried

  1. Development and validation of an immunohistochemical method for rapid diagnosis of swine erysipelas in formalin-fixed, paraffin-embedded tissue samples.

    PubMed

    Opriessnig, Tanja; Bender, Joseph S; Halbur, Patrick G

    2010-01-01

    The objective of the study was to develop an immunohistochemical (IHC) assay for rapid detection of Erysipelothrix rhusiopathiae. Serotypes 1a, 1b, and 2 are most frequently associated with clinical disease in pigs. Antiserum against serotypes 1a, 1b, and 2 was produced in rabbits, pooled, and applied to formalin-fixed, paraffin-embedded tissue sections of pigs (lungs, heart, spleen, and skin). The results obtained with the IHC assay were compared with direct culture on tissue samples from experimentally inoculated pigs either treated (n = 6) with antibiotics or untreated (n = 8) as well as on samples from field cases (n = 170) submitted to the Veterinary Diagnostic Laboratory at Iowa State University. The agreement between direct culture and IHC staining was found to be substantial. The results of the present study indicate that the IHC assay is highly sensitive and specific in detecting E. rhusiopathiae antigen in formalin-fixed, paraffin-embedded tissues. Results indicated that the IHC is particularly useful in cases in which pigs had been treated with antibiotics prior to submission and in which direct cultures of organs were negative. In addition, the IHC was found to be useful for detection of E. rhusiopathiae antigen in skin lesions, which are often culture negative.

  2. Incidence of mycobacterial infections in cats in Great Britain: estimate from feline tissue samples submitted to diagnostic laboratories.

    PubMed

    Gunn-Moore, D A; Gaunt, C; Shaw, D J

    2013-08-01

    The aim of this study was to estimate the incidence of mycobacterial infections in cats in Great Britain (GB). This was performed using the proxy measure of feline tissue samples submitted to diagnostic laboratories in GB that were found to have histopathological changes typical of mycobacterial infection ('MYC'). Sixteen primary diagnostic laboratories were asked for information on the number of feline samples submitted in 2009, the number with MYC, the number undergoing Ziehl-Neelsen (ZN) staining and, for comparison, the number diagnosed with lymphoma. Eight laboratories provided full data for the whole year: 11,782 samples; lymphoma 3.2% (mean, 95% CI: 2.89, 3.5), MYC 1.16% (0.98; 1.37) and ZN-positive 0.31% (0.22; 0.43). Data on 1569 samples from seven laboratories that provided partial data on samples for the whole year revealed similar results, although all changes were more frequent: lymphoma 5.42% (4.35; 6.66), MYC 2.36% (1.66; 3.23) and ZN-positive 0.77% (0.40; 1.33). One laboratory only provided data for part of the year (4.5 months), reporting all three types of histopathology less frequently: 18,232 samples; lymphoma 0.2% (0.18; 0.32), MYC 0.07% (0.04; 0.12) and ZN-positive 0.05% (0.02; 0.09). The reasons for low reporting rates in this high-throughput laboratory are unclear. In total, 187 samples were reported as having MYC. Five Reference laboratories were also contacted, reporting 174 feline tissue submissions in 2009, with mycobacteria being cultured from 90. The study shows that MYC are frequently reported in tissue samples from cats in GB, being reported in ~1% of samples, with confirmation as ZN-positive in ~0.3%. Lymphoma is recognized as a common disease in cats, being seen in ~3% of samples in this study. When compared against MYC, lymphoma was reported only twice as frequently. This confirms that far from being rare, clinically significant mycobacterial infections occur commonly in cats in GB.

  3. Predictors of victim disclosure in child sexual abuse: Additional evidence from a sample of incarcerated adult sex offenders.

    PubMed

    Leclerc, Benoit; Wortley, Richard

    2015-05-01

    The under-reporting of child sexual abuse by victims is a serious problem that may prolong the suffering of victims and leave perpetrators free to continue offending. Yet empirical evidence indicates that victim disclosure rates are low. In this study, we perform regression analysis with a sample of 369 adult child sexual offenders to examine potential predictors of victim disclosure. Specifically, we extend the range of previously examined potential predictors of victim disclosure and investigate interaction effects in order to better capture under which circumstances victim disclosure is more likely. The current study differs from previous studies in that it examines the impact of victim and offense variables on victim disclosure from the perspective of the offender. In line with previous studies, we found that disclosure increased with the age of the victim and if penetration had occurred. In addition, we found that disclosure increased when the victim came from a non-dysfunctional family and resisted the abuse. The presence of an interaction effect highlighted the impact of the situation on victim disclosure. This effect indicated that as victims get older, they are more likely to disclose the abuse when they are not living with the offender at the time of abuse, but less likely to do so when they are living with the offender at the time of abuse. These findings are discussed in relation to previous studies and the need to facilitate victim disclosure.

  4. A low dimensional entropy-based descriptor of several tissues in skin cancer histopathology samples

    NASA Astrophysics Data System (ADS)

    Álvarez, Pablo; Corredor, Germán.; García-Arteaga, Juan D.; Romero, Eduardo

    2015-12-01

    The use of low-level visual features to assign high level labels in datasets of histopathology images is a possible solution to the problems derived from manual labeling by experts. However, in many cases, the visual cues are not enough. In this article we propose the use of features derived exclusively from the spatial distribution of the cell nuclei. These features are calculated using the weight of k-nn graphs constructed from the distances between cells. Results show that there are k values with enhanced discriminatory power, especially when comparing cancerous and non-cancerous tissue.

  5. Comparing paraffined and deparaffinized breast cancer tissue samples and an analysis of Raman spectroscopy and infrared methods

    NASA Astrophysics Data System (ADS)

    Depciuch, J.; Kaznowska, E.; Szmuc, K.; Zawlik, I.; Cholewa, M.; Heraud, P.; Cebulski, J.

    2016-05-01

    Breast cancer makes up a quarter of all cancer in women, which is why research into new diagnostic methods and sample preparations need to be developed at an accelerated pace. Researchers are looking for diagnostic tools to detect when an individual has cancer cells and use that information to see what measurements and approaches can be used to take further diagnostic steps. The most common method of sample preparation is the imbibing of tumor tissue in paraffin, which can produce a background for spectroscopic measurements in the range of 500-3500 cm-1. In this study we demonstrated that proper preparation of paraffin-embedded specimens and the measurement methodology can eliminate paraffin vibration, as was done in the work Depciuch et al. 2015. Thanks to this spectroscopic technique there may become a reliable and accurate method of diagnosing breast cancer based on the evidence found from the prepared samples. The study compared the results obtained through Raman spectroscopy and FTIR (Fourier Transform Infrared) measurements of healthy and cancerous breast tissues that were either embedded in paraffin or deparaffinized. The resulting spectrum and accurate analysis led to the conclusion that the appropriate measurement of the background and the elimination of peaks from the paraffin had the greatest impact on the reliability of results. Furthermore, after the accurate, detailed studies FTIR and Raman spectroscopy on samples of breast tissue that were deparaffinized or embedded in paraffin, including a complete analysis of the peak after transformation Kramers-Kröning (KK), it was found that sample preparation did not affect the result obtained by measuring the reflectance in the mid-infrared range, and that this only had a minimal effect relating to the intensity obtained by the measurement of the Raman peak. Only in special cases, when Raman spectroscopic methods are used for research to find the peculiarities of the spectra, are deparaffinization recommended

  6. 3-Dimensional quantitative detection of nanoparticle content in biological tissue samples after local cancer treatment

    NASA Astrophysics Data System (ADS)

    Rahn, Helene; Alexiou, Christoph; Trahms, Lutz; Odenbach, Stefan

    2014-06-01

    X-ray computed tomography is nowadays used for a wide range of applications in medicine, science and technology. X-ray microcomputed tomography (XμCT) follows the same principles used for conventional medical CT scanners, but improves the spatial resolution to a few micrometers. We present an example of an application of X-ray microtomography, a study of 3-dimensional biodistribution, as along with the quantification of nanoparticle content in tumoral tissue after minimally invasive cancer therapy. One of these minimal invasive cancer treatments is magnetic drug targeting, where the magnetic nanoparticles are used as controllable drug carriers. The quantification is based on a calibration of the XμCT-equipment. The developed calibration procedure of the X-ray-μCT-equipment is based on a phantom system which allows the discrimination between the various gray values of the data set. These phantoms consist of a biological tissue substitute and magnetic nanoparticles. The phantoms have been studied with XμCT and have been examined magnetically. The obtained gray values and nanoparticle concentration lead to a calibration curve. This curve can be applied to tomographic data sets. Accordingly, this calibration enables a voxel-wise assignment of gray values in the digital tomographic data set to nanoparticle content. Thus, the calibration procedure enables a 3-dimensional study of nanoparticle distribution as well as concentration.

  7. Tissue-Specific DNA Methylation Patterns in Forensic Samples Detected by Pyrosequencing®.

    PubMed

    Antunes, Joana; Balamurugan, Kuppareddi; Duncan, George; McCord, Bruce

    2015-01-01

    In certain circumstances the outcome of a trial may hinge on the ability of a forensic laboratory to determine the identity of biological stains present at crime scenes. An example of such a situation would be the detection of blood, saliva, vaginal fluid, or other body fluid in a specific location whereby its presence would reinforce the victim's or suspect's version of the events that happened during the commission of a crime. However, current serological methods used for identifying body fluids may lack the sensitivity and specificity to identify these fluids, particularly for trace levels. New procedures using proteomic methods and RNA-based gene expression show promise in addressing this issue; however, concerns about stability and relative levels of gene expression remain. An alternative approach is to utilize patterns of epigenetic DNA methylation. DNA methylation is an epigenetic mechanism that regulates the specificity of genes being expressed or silenced in cells. Regions in the human genome referred to as tissue-specific differentially methylated regions account for unique patterns of DNA methylation that are specific for each cell type. This chapter addresses the application of bisulfite-modified PCR combined with Pyrosequencing(®) to detect tissue-specific DNA methylation patterns and perform trace serological analysis. The quantitative nature and precision available with Pyrosequencing presents major advantages in these studies as it permits detection of and contrast between cells with differential levels of methylation. The procedure can be applied to a variety of biological fluids which may be present at crime scenes.

  8. Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: Focus on sample preparation and derivatization conditions.

    PubMed

    Weber, Daniela; Davies, Michael J; Grune, Tilman

    2015-08-01

    Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented for the spectrophotometric and HPLC method as well as for immunoblotting and ELISA. An extensive overview covering these methods in previously published articles is given for researchers who plan to measure protein carbonyls in different samples.

  9. Evidence of presence of Mycobacterium tuberculosis in bovine tissue samples by multiplex PCR: possible relevance to reverse zoonosis.

    PubMed

    Mittal, M; Chakravarti, S; Sharma, V; Sanjeeth, B S; Churamani, C P; Kanwar, N S

    2014-04-01

    Bovine tuberculosis, caused by Mycobacterium bovis, remains one of the most important zoonotic health concerns worldwide. The transmission of Mycobacterium tuberculosis from humans to animals also occurs especially in countries where there is close interaction of humans with the animals. In the present study, thirty bovine lung tissue autopsy samples from an organized dairy farm located in North India were screened for the presence of Mycobacterium tuberculosis complex by smear microscopy, histopathological findings and PCR. Differential diagnosis of M. tuberculosis and M. bovis was made based on the deletion of mce-3 operon in M. bovis. The present study found eight of these samples positive for M. tuberculosis by multiplex PCR. Sequencing was performed on two PCR-positive representative samples and on annotation, and BLAST analysis confirmed the presence of gene fragment specific to Mycobacterium tuberculosis. The presence of M. tuberculosis in all the positive samples raises the possibility of human-to-cattle transmission and possible adaptation of this organism in bovine tissues. This study accentuates the importance of screening and differential diagnosis of Mycobacterium tuberculosis complex in humans and livestock for adopting effective TB control and eradication programmes.

  10. Papillary reconstruction and guided tissue regeneration for combined periodontal-endodontic lesions caused by palatogingival groove and additional root: a case report.

    PubMed

    Miao, Hui; Chen, Min; Otgonbayar, Tsetsen; Zhang, Sha Sha; Hou, Min Hong; Wu, Zhou; Wang, Yong Lan; Wu, Li Geng

    2015-12-01

    We described a combined periodontal-endodontic lesion, which was caused by a palatogingival groove and an additional root. An interdisciplinary approach involving endodontic therapy, mineral trioxide aggregate (MTA) filling, root resection, guided tissue regeneration, and papillary reconstruction was used for the case. The tooth presents morphologically and functionally normal except tooth discoloration caused by MTA.

  11. An efficient protocol for tissue sampling and DNA isolation from the stem bark of Leguminosae trees.

    PubMed

    Novaes, R M L; Rodrigues, J G; Lovato, M B

    2009-02-03

    Traditionally, molecular studies of plant species have used leaves as the source of DNA. However, sampling leaves from tall tree species can be quite difficult and expensive. We developed a sequence of procedures for using stem bark as a source of DNA from Leguminosae trees of the Atlantic Forest and the Cerrado. Leguminosae is an important species-rich family in these two highly diverse and endangered biomes. A modified CTAB protocol for DNA isolation is described, and details of the procedures for sampling and storage of the bark are given. The procedures were initially developed for three species, and then their applicability for 15 other species was evaluated. DNA of satisfactory quality was obtained from the bark of all species. The amounts of DNA obtained from leaves were slightly higher than from bark samples, while its purity was the same. Storing the bark frozen or by drying in silica gel yielded similar results. Polymerase chain reaction amplification worked for both plastid and nuclear genomes. This alternative for isolating DNA from bark samples of trees facilitates field work with these tree species.

  12. Determination of five nitroimidazole residues in artificial porcine muscle tissue samples by capillary electrophoresis.

    PubMed

    Lin, Yingyun; Su, Yan; Liao, Xiulin; Yang, Na; Yang, Xiupei; Choi, Martin M F

    2012-01-15

    A capillary electrophoresis (CE) method with ultraviolet detection has been developed for simultaneous detection and quantification of five nitroimidazoles including benzoylmetronidazole, dimetridazole, metronidazole, ronidazole, and secnidazole in porcine muscles. Nitroimidazoles in samples were extracted by ethyl acetate with subsequent clean-up by a strong cation exchange solid phase extraction column. The clean extracts were subjected to CE separation with optimal experimental conditions: pH 3.0 running buffer containing 25mM sodium phosphate and 0.10mM tetrabutylammonium bromide, 5s hydrodynamic injection at 0.5psi and 28kV separation voltage. The nitroimidazoles could be monitored and detected at 320nm within 18min. The limits of detection were below 1.0μg/kg and limits of quantification were lower than 3.2μg/kg for all nitroimidazoles in the muscle samples. The recoveries and relative standard deviations were 85.4-96.0, 83.5-92.5, 1.3-3.9, and 1.1-4.2%, respectively for the intra-day and inter-day analyses. The proposed CE method has been successfully applied to determine nitroimidazoles in artificial porcine muscle samples with good accuracy and recovery, demonstrating that it has potential for detection and quantification of multi-nitroimidazole residue in real muscle samples.

  13. Comparing elastographic strain images with modulus images obtained using nanoindentation: preliminary results using phantoms and tissue samples.

    PubMed

    Srinivasan, Seshadri; Krouskop, T; Ophir, Jonathan

    2004-03-01

    Conventional elastography involves quasistatic mechanical compression (external or internal) of the tissue under ultrasonic insonification to obtain radiofrequency (RF) A-lines before and after compression. Cross-correlation of the pre- and postcompression A-lines results in displacement images with axial gradients that produce the strain images (strain elastograms). Though the strain elastograms show structural similarities to the modulus images, they are not related in a simple way to the modulus images because the strains depend on both modulus and geometry of the materials being deformed. Therefore, a quantification of the similarities between the strain and modulus images may enhance the interpretation confidence of strain elastograms in depicting tissue structure. To demonstrate similarities between modulus images and strain elastograms, a feasibility study of using nanoindentation to obtain modulus images of thin slices of tissue and tissue-mimicking phantoms (agar-gelatin mixtures) was performed first, with encouraging results. This was followed by a comparison of modulus images and strain elastograms obtained from the same sample slices. The experimental results indicated that, under certain experimental conditions, it is feasible to perform quantitative comparisons between strain images (using elastography) and modulus images. A good visual, as well as quantitative, correspondence between structures in the modulus and strain images could be obtained at a 3-mm scale.

  14. Biodistribution Analysis of Oncolytic Adenoviruses in Patient Autopsy Samples Reveals Vascular Transduction of Noninjected Tumors and Tissues

    PubMed Central

    Koski, Anniina; Bramante, Simona; Kipar, Anja; Oksanen, Minna; Juhila, Juuso; Vassilev, Lotta; Joensuu, Timo; Kanerva, Anna; Hemminki, Akseli

    2015-01-01

    In clinical trials with oncolytic adenoviruses, there has been no mortality associated with treatment vectors. Likewise, in the Advanced Therapy Access Program (ATAP), where 290 patients were treated with 10 different viruses, no vector-related mortality was observed. However, as the patient population who received adenovirus treatments in ATAP represented heavily pretreated patients, often with very advanced disease, some patients died relatively soon after receiving their virus treatment mandating autopsy to investigate cause of death. Eleven such autopsies were performed and confirmed disease progression as the cause of death in each case. The regulatory requirement for investigating the safety of advanced therapy medical products presented a unique opportunity to study tissue samples collected as a routine part of the autopsies. Oncolytic adenoviral DNA was recovered in a wide range of tissues, including injected and noninjected tumors and various normal tissues, demonstrating the ability of the vector to disseminate through the vascular route. Furthermore, we recovered and cultured viable virus from samples of noninjected brain metastases of an intravenously treated patient, confirming that oncolytic adenovirus can reach tumors through the intravascular route. Data presented here give mechanistic insight into mode of action and biodistribution of oncolytic adenoviruses in cancer patients. PMID:26156245

  15. Substrate contributions in micro-ATR of thin samples: implications for analysis of cells, tissue and biological fluids.

    PubMed

    Bassan, Paul; Sachdeva, Ashwin; Lee, Joe; Gardner, Peter

    2013-07-21

    Low-e microscope slides are a common substrate for biological samples. Typically they are used for transflection infrared microspectroscopy but increasingly they are also being used for micro-ATR experiments since it is assumed that the FTIR-ATR absorbance spectra of cells and tissue on low-e substrates will not contain any spectral contributions from the substrate materials. This, in part, is due to the expectation that all the infrared light will be reflected at the highly reflective surface. At low sample thicknesses, however (e.g. less than 2 μm) the electric field does indeed penetrate through the substrate layers and undergoes absorption, from the glass supporting layer making up the majority of the slide. In this paper we show experimental evidence of the substrate contributions in ATR spectra and also a theoretical model giving insight into the spectral contributions of the substrate as a function of sample thickness.

  16. Development of a chromogenic in situ hybridization for Giardia duodenalis and its application in canine, feline, and porcine intestinal tissues samples

    PubMed Central

    Weissenböck, Herbert; Ondrovics, Martina; Gurtner, Susanne; Schiessl, Peter; Mostegl, Meike M.; Richter, Barbara

    2011-01-01

    In the present study, a chromogenic in situ hybridization for the identification of Giardia duodenalis in paraffin-embedded tissue samples was developed. The sensitivity and specificity of the probe was validated by testing it on cultured reference samples of different assemblages of G. duodenalis as well as culture and tissue samples containing other protozoa and infectious agents. The probe gave a positive reaction with the Giardia samples and a negative reaction with all other samples. Further, the probe was used for screening of histological slides of intestine from different animal species (99 canine samples, 85 feline samples, and 202 porcine samples) for the presence of G. duodenalis trophozoites. With this assay, the parasites were detected in samples from 8 dogs (8.08%), 6 cats (7.06%), and zero pigs. The results clearly indicate that the described method is useful for detection of Giardia trophozoites in routinely processed intestinal tissue of different animal species. PMID:21908276

  17. Pharmacokinetic study on pradofloxacin in the dog – Comparison of serum analysis, ultrafiltration and tissue sampling after oral administration

    PubMed Central

    2013-01-01

    Background Pradofloxacin, a newly developed 8-cyano-fluoroquinolone, show enhanced activity against Gram-positive organisms and anaerobes to treat canine and feline bacterial infections. The purpose of this cross-over study was to measure the unbound drug concentration of pradofloxacin in the interstitial fluid (ISF) using ultrafiltration and to compare the kinetics of pradofloxacin in serum, ISF and tissue using enrofloxacin as reference. Results After oral administration of enrofloxacin (5 mg/kg) and pradofloxacin (3 mg/kg and 6 mg/kg, respectively), serum collection and ultrafiltration in regular intervals over a period of 24 h were performed, followed by tissue sampling at the end of the third dosing protocol (pradofloxacin 6 mg/kg). Peak concentrations of pradofloxacin (3 mg/kg) were 1.55±0.31 μg/ml in the ISF and 1.85±0.23 μg/ml in serum and for pradofloxacin (6 mg/kg) 2.71±0.81 μg/kg in the ISF and 2.77±0.64 μg/kg in serum; both without a statistical difference between ISF and serum. Comparison between all sampling approaches showed no consistent pattern of statistical differences. Conclusions Despite some technical shortcomings the ultrafiltration approach appears to be the most sensitive sampling technique to estimate pharmacokinetic values of pradofloxacin at the infection site. Pharmacokinetics – Pradofloxacin – Ultrafiltration – Dog – Oral Administration. PMID:23410255

  18. Non-lethal sampling of liver tissue for toxicologic evaluation of Florida cottonmouths snakes, Agkistrodon piscivorus conanti.

    PubMed

    Quesada, Rolando J; McCleary, Ryan J R; Heard, Darryl J; Lillywhite, Harvey B

    2014-01-01

    Due to their longevity, strong site tenure, poikilothermic metabolism, and low-energy specializations, reptiles might serve as excellent environmental sentinels. Cottonmouth snakes are generalist predators and scavengers, and as such, may have higher exposure to persistent environmental contaminants as a result of bioaccumulation. Traditionally, assessment and monitoring of contaminant exposure in reptiles have involved lethal sampling techniques. In this paper, we describe a non-destructive technique for sampling liver tissue in live anesthetized Florida cottonmouths. Wild-caught snakes (n = 21) were anesthetized with propofol, and a liver wedge biopsy was obtained by clamping the edge of the organ with two small hemostatic mosquito forceps via right-sided coeliotomy incision. A minimum required tissue sample weighing >100 mg was harvested from all except one of the animals. No mortalities occurred during the procedures or recovery from anesthesia, and all snakes were released back into the field after the animal had consumed prey and defecated, usually within 2 weeks following surgery. Hemorrhage was a minor complication in most snakes, especially those with friable discolored livers. The procedure appeared to have no short-term deleterious effects, and two biopsied individuals were captured after being released into the field and appeared to be normal and healthy. However, follow-up studies and recapture of more snakes are needed to assess long-term survivability. Our non-destructive liver sampling technique might be implemented in toxicological studies of other squamates and could help to minimize the lethal sampling of threatened species.

  19. Diagnostic utility of immunohistochemistry in distinguishing trichoepithelioma and basal cell carcinoma: evaluation using tissue microarray samples.

    PubMed

    Tebcherani, Antonio José; de Andrade, Heitor Franco; Sotto, Mirian N

    2012-10-01

    Trichoepithelioma is a benign neoplasm that shares both clinical and histological features with basal cell carcinoma. It is important to distinguish these neoplasms because they require different clinical behavior and therapeutic planning. Many studies have addressed the use of immunohistochemistry to improve the differential diagnosis of these tumors. These studies present conflicting results when addressing the same markers, probably owing to the small number of basaloid tumors that comprised their studies, which generally did not exceed 50 cases. We built a tissue microarray with 162 trichoepithelioma and 328 basal cell carcinoma biopsies and tested a panel of immune markers composed of CD34, CD10, epithelial membrane antigen, Bcl-2, cytokeratins 15 and 20 and D2-40. The results were analyzed using multiple linear and logistic regression models. This analysis revealed a model that could differentiate trichoepithelioma from basal cell carcinoma in 36% of the cases. The panel of immunohistochemical markers required to differentiate between these tumors was composed of CD10, cytokeratin 15, cytokeratin 20 and D2-40. The results obtained in this work were generated from a large number of biopsies and resulted in the confirmation of overlapping epithelial and stromal immunohistochemical profiles from these basaloid tumors. The results also corroborate the point of view that trichoepithelioma and basal cell carcinoma tumors represent two different points in the differentiation of a single cell type. Despite the use of panels of immune markers, histopathological criteria associated with clinical data certainly remain the best guideline for the differential diagnosis of trichoepithelioma and basal cell carcinoma.

  20. Progression from high insulin resistance to type 2 diabetes does not entail additional visceral adipose tissue inflammation.

    PubMed

    Barbarroja, Nuria; Lopez-Pedrera, Chary; Garrido-Sanchez, Lourdes; Mayas, Maria Dolores; Oliva-Olivera, Wilfredo; Bernal-Lopez, Maria Rosa; El Bekay, Rajaa; Tinahones, Francisco Jose

    2012-01-01

    Obesity is associated with a low-grade chronic inflammation state. As a consequence, adipose tissue expresses pro-inflammatory cytokines that propagate inflammatory responses systemically elsewhere, promoting whole-body insulin resistance and consequential islet β-cell exhaustation. Thus, insulin resistance is considered the early stage of type 2 diabetes. However, there is evidence of obese individuals that never develop diabetes indicating that the mechanisms governing the association between the increase of inflammatory factors and type 2 diabetes are much more complex and deserve further investigation. We studied for the first time the differences in insulin signalling and inflammatory pathways in blood and visceral adipose tissue (VAT) of 20 lean healthy donors and 40 equal morbidly obese (MO) patients classified in high insulin resistance (high IR) degree and diabetes state. We studied the changes in proinflammatory markers and lipid content from serum; macrophage infiltration, mRNA expression of inflammatory cytokines and transcription factors, activation of kinases involved in inflammation and expression of insulin signalling molecules in VAT. VAT comparison of these experimental groups revealed that type 2 diabetic-MO subjects exhibit the same pro-inflammatory profile than the high IR-MO patients, characterized by elevated levels of IL-1β, IL-6, TNFα, JNK1/2, ERK1/2, STAT3 and NFκB. Our work rules out the assumption that the inflammation should be increased in obese people with type 2 diabetes compared to high IR obese. These findings indicate that some mechanisms, other than systemic and VAT inflammation must be involved in the development of type 2 diabetes in obesity.

  1. Progression from High Insulin Resistance to Type 2 Diabetes Does Not Entail Additional Visceral Adipose Tissue Inflammation

    PubMed Central

    Barbarroja, Nuria; Lopez-Pedrera, Chary; Garrido-Sanchez, Lourdes; Mayas, Maria Dolores; Oliva-Olivera, Wilfredo; Bernal-Lopez, Maria Rosa; El Bekay, Rajaa; Tinahones, Francisco Jose

    2012-01-01

    Obesity is associated with a low-grade chronic inflammation state. As a consequence, adipose tissue expresses pro-inflammatory cytokines that propagate inflammatory responses systemically elsewhere, promoting whole-body insulin resistance and consequential islet β-cell exhaustation. Thus, insulin resistance is considered the early stage of type 2 diabetes. However, there is evidence of obese individuals that never develop diabetes indicating that the mechanisms governing the association between the increase of inflammatory factors and type 2 diabetes are much more complex and deserve further investigation. We studied for the first time the differences in insulin signalling and inflammatory pathways in blood and visceral adipose tissue (VAT) of 20 lean healthy donors and 40 equal morbidly obese (MO) patients classified in high insulin resistance (high IR) degree and diabetes state. We studied the changes in proinflammatory markers and lipid content from serum; macrophage infiltration, mRNA expression of inflammatory cytokines and transcription factors, activation of kinases involved in inflammation and expression of insulin signalling molecules in VAT. VAT comparison of these experimental groups revealed that type 2 diabetic-MO subjects exhibit the same pro-inflammatory profile than the high IR-MO patients, characterized by elevated levels of IL-1β, IL-6, TNFα, JNK1/2, ERK1/2, STAT3 and NFκB. Our work rules out the assumption that the inflammation should be increased in obese people with type 2 diabetes compared to high IR obese. These findings indicate that some mechanisms, other than systemic and VAT inflammation must be involved in the development of type 2 diabetes in obesity. PMID:23110196

  2. Molecular identification of human tuberculosis in recent and historic bone tissue samples: The role of molecular techniques for the study of historic tuberculosis.

    PubMed

    Zink, Albert R; Grabner, Waltraud; Nerlich, Andreas G

    2005-01-01

    We describe the molecular identification of the M. tuberculosis complex DNA in bone tissue samples from recent and historic populations. In a first set, archival paraffin material from vertebral bodies of 12 recent cases with clinically/microbiologically proven tuberculosis was compared to 12 further cases without tuberculosis. While eight TB cases revealed a specific mycobacterial amplification product, none of the controls was positive. Interestingly, one case with tuberculous sepsis (Landouzy sepsis), five cases with tuberculous spread beyond the primarily affected organ (i.e., lymph node or miliar involvement), and also two of six cases with restricted pulmonary tuberculosis reacted positively in the vertebral specimens. This indicates that a molecular analysis can detect mycobacteria even in unremarkable bone tissue, proving that organ tuberculosis is present. In addition, the extent of spread is of high significance for the frequency of positive reactions. In addition, we investigated a series of vertebral samples coming from an Egyptian population of the necropolis of Thebes-West dating to approximately 1450-500 BC. In this group of 36 cases, three of five cases with typical macromorphological signs for tuberculous spondylitis, 2 of 12 cases with nonspecific alterations, and 2 of 19 cases without macroscopic pathology revealed a specific amplicon of the M. tuberculosis complex. This suggests a significant frequency of infected people in that ancient population. Finally, a fourth group of 51 long bone samples with pathological alterations coming form a southern German ossuary (between AD 1400-1800) was investigated, and 10 cases were positive for the M. tuberculosis complex. These studies of historic material clearly support the notion that tuberculous infections can be unequivocally identified by molecular techniques. The relatively high frequency of ancient bacterial DNA amplifications in unremarkable bone is well-explained by our analysis of the recent

  3. Development of a remote laser-induced breakdown spectroscopy system for investigation of calcified tissue samples

    SciTech Connect

    Hrdlicka, Ales; Prokes, Lubomir; Stankova, Alice; Novotny, Karel; Vitesnikova, Anna; Kanicky, Viktor; Otruba, Vitezslav; Kaiser, Jozef; Novotny, Jan; Malina, Radomir; Palenikova, Katerina

    2010-05-01

    The development of a remote laser-induced breakdown spectroscopy (LIBS) setup with an off-axis Newtonian collection optics, Galilean-based focusing telescope, and a 532 nm flattop laser beam source is presented. The device was tested at a 6 m distance on a slice of bone to simulate its possible use in the field, e.g., during archaeological excavations. It is shown that this setup is sufficiently sensitive to both major (P, Mg) and minor elements (Na, Zn, Sr). The measured quantities of Mg, Zn, and Sr correspond to the values obtained by reference laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) measurements within an approximately 20% range of uncertainty. A single point calibration was performed by use of a bone meal standard . The radial element distribution is almost invariable by use of LA-ICP-MS, whereas the LIBS measurement showed a strong dependence on the sample porosity. Based on these results, this remote LIBS setup with a relatively large (350 mm) collecting mirror is capable of semiquantitative analysis at the level of units of mg kg{sup -1}.

  4. Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures

    PubMed Central

    Ou, Yangguang; Wu, Juanfang; Sandberg, Mats

    2014-01-01

    This review covers recent advances in sampling fluid from the extracellular space of brain tissue by electroosmosis (EO). Two techniques, EO sampling with a single fused-silica capillary and EO push–pull perfusion, have been developed. These tools were used to investigate the function of membrane-bound enzymes with outward-facing active sites, or ectoenzymes, in modulating the activity of the neuropeptides leu-enkephalin and galanin in organotypic-hippocampal-slice cultures (OHSCs). In addition, the approach was used to determine the endogenous concentration of a thiol, cysteamine, in OHSCs. We have also investigated the degradation of coenzyme A in the extracellular space. The approach provides information on ectoenzyme activity, including Michaelis constants, in tissue, which, as far as we are aware, has not been done before. On the basis of computational evidence, EO push–pull perfusion can distinguish ectoenzyme activity with a ~100 µm spatial resolution, which is important for studies of enzyme kinetics in adjacent regions of the rat hippocampus. PMID:25168111

  5. The problem with medical research on tissue and organ samples taken in connection with forensic autopsies in France.

    PubMed

    Rougé-Maillart, C; Dupont, V; Jousset, N

    2016-02-01

    Currently, in France, it is legally impossible to conduct scientific research on tissue and organ samples taken from forensic autopsies. In fact, the law schedules the destruction of such samples at the end of the judicial investigation, and the common law rules governing cadaver research cannot be applied to the forensic context. However, nothing seems in itself to stand in the way of such research since, despite their specific nature, these samples from forensic autopsies could be subject, following legislative amendments, to common law relating to medical research on samples taken from deceased persons. But an essential legislative amendment, firstly to allow the Biomedicine Agency to become authorized to issue a research permit and secondly, to change the research conditions in terms of the non-opposition of the deceased to said research. Such an amendment would be a true breakthrough because it would allow teams to continue to move forward calmly in research, and allow this research to be placed within a legal framework, which would promote international exchanges.

  6. A minimally invasive method of piscine tissue collection and an analysis of long-term field-storage conditions for samples

    PubMed Central

    Campanella, James J; Smalley, John V

    2006-01-01

    Background The acquisition of high-quality DNA for use in phylogenetic and molecular population genetic studies is a primary concern for evolutionary and genetic researchers. Many non-destructive DNA sampling methods have been developed and are used with a variety of taxa in applications ranging from genetic stock assessment to molecular forensics. Results The authors have developed a field sampling method for obtaining high-quality DNA from sunfish (Lepomis) and other freshwater fish that employs a variation on the buccal swab method and results in the collection of DNA suitable for PCR amplification and polymorphism analysis. Additionally, since the circumstances of storage are always a concern for field biologists, the authors have tested the potential storage conditions of swabbed samples and whether those conditions affect DNA extraction and PCR amplification. It was found that samples stored at room temperature in the dark for over 200 days could still yield DNA suitable for PCR amplification and polymorphism detection. Conclusion These findings suggest that valuable molecular genetic data may be obtained from tissues that have not been treated or stored under optimal field conditions. Furthermore, it is clear that the lack of adequately low temperatures during transport and long term storage should not be a barrier to anyone wishing to engage in field-based molecular genetic research. PMID:16734898

  7. Effect of addition of antibiotics and an antioxidant on the stability of tissue reference materials for domoic acid, the amnesic shellfish poison.

    PubMed

    McCarron, Pearse; Burrell, Stephen; Hess, Philipp

    2007-04-01

    Five separate reference materials (RMs) were prepared from a mussel (Mytilus edulis) tissue containing domoic acid (DA) from scallop hepatopancreas (Pecten maximus). Homogenates were separately spiked with antibiotics, an antioxidant, or a combination of both. Control materials did not contain any additives and were prepared from lightly cooked and autoclaved mussel tissues. Stability studies were run over a 148-day period at three different temperature conditions: -20 degrees C, +4 degrees C and +40 degrees C. DA contents in all materials were characterised by HPLC-UV. Homogeneities were demonstrated at the beginning of the study, with coefficients of variance of less than 4% (n = 9). DA was stable at -20 degrees C in all materials. The control materials showed significant degradation after two days at +40 degrees C, and after eight days at +4 degrees C. Each of the materials containing additives demonstrated better stability during the initial period of the study. In addition there was no significant degradation in any of the materials with additives stored at +4 degrees C over the duration of the study. The material containing a combination of the antibiotics and the antioxidant displayed the best stability of all the materials. There was no significant reduction in DA concentration at all temperature conditions after eight days, and after 32 days the decrease at +40 degrees C was still <20 %. Following this, a DA laboratory reference material (LRM) was prepared and, based on previous results, spiked with both the antioxidant and antibiotics. A short-term stability study on this material gave similar results to the corresponding material in the additives study. This study shows that combined use of the additives investigated in the preparation of a mussel tissue reference material for DA ensures analyte stability for a period of up to eight days at temperatures of up to +40 degrees C, a condition that is particularly important when shipping test materials

  8. The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.

    PubMed

    Cai, Jing; Li, Tao; Huang, Bangxing; Cheng, Henghui; Ding, Hui; Dong, Weihong; Xiao, Man; Liu, Ling; Wang, Zehua

    2014-01-01

    Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.

  9. Distribution of Mycobacterium avium Complex Isolates in Tissue Samples of Pigs Fed Peat Naturally Contaminated with Mycobacteria as a Supplement

    PubMed Central

    Matlova, Ludmila; Dvorska, Lenka; Ayele, Wuhib Yayo; Bartos, Milan; Amemori, Takashi; Pavlik, Ivo

    2005-01-01

    In early 1999, there was an increased incidence of tuberculous lesions in the lymph nodes of slaughtered pigs in the Czech Republic. In part 1 of this study, tuberculous lesions were detected in 140 (62%) tissue samples collected from pigs coming from 15 farms in 15 districts at routine veterinary meat inspections in abattoirs. Mycobacteria were isolated from 37 (16%) tissue samples: 34 Mycobacterium avium subsp. hominissuis isolates and three environmentally derived mycobacteria. In search of infection sources, M. avium subsp. hominissuis was isolated from 38 (79%) samples of peat used as a feed supplement. In part 2 of our study, the head, mesenteric, and inguinal lymph nodes of 117 randomly selected slaughtered pigs from one farm with young piglets fed peat as a supplement were investigated for mycobacterial infection. From 65 (56%) pigs, a total of 76 mycobacterial isolates were identified (56 M. avium subsp. hominissuis isolates, 5 M. avium subsp. avium isolates, 3 M. intracellulare isolates, and 12 environmentally derived mycobacterial isolates). IS1245 restriction fragment length polymorphism (RFLP) types with >20 bands of 45 distinct RFLP types were found in 49 M. avium subsp. hominissuis isolates from pigs (n = 31) and peat (n = 18). Identical RFLP types were found in only four pig isolates. Five randomly selected isolates from pigs and peat were subcultured to six independent clones or colonies. Among the IS1245 RFLP types of 30 clones, identical RFLP types obtained from pigs and peat were identified, which confirmed the hypothesis that peat contaminated with mycobacteria represents a significant source of mycobacterial infection for pigs. PMID:15750094

  10. Detection of triazole deicing additives in soil samples from airports with low, mid, and large volume aircraft deicing activities.

    PubMed

    McNeill, K S; Cancilla, D A

    2009-03-01

    Soil samples from three USA airports representing low, mid, and large volume users of aircraft deicing fluids (ADAFs) were analyzed by LC/MS/MS for the presence of triazoles, a class of corrosion inhibitors historically used in ADAFs. Triazoles, specifically the 4-methyl-1H-benzotriazole and the 5-methyl-1H-benzotriazole, were detected in a majority of samples and ranged from 2.35 to 424.19 microg/kg. Previous studies have focused primarily on ground and surface water impacts of larger volume ADAF users. The detection of triazoles in soils at low volume ADAF use airports suggests that deicing activities may have a broader environmental impact than previously considered.

  11. Electrofishing and the effects of depletion sampling on fish health: A review and recommendations for additional study

    USGS Publications Warehouse

    Panek, F.M.; Densmore, Christine L.; Cipriano, R.C.; Bruckner, A.W.; Shchelkunov, I.S.

    2011-01-01

    Depletion sampling in combination with multiple-pass electrofishing is an important fisheries management tool for wadeable streams. This combination of techniques has been used routinely by federal and state fishery management agencies for several decades as a reliable means to obtain quantitative data on trout populations or to describe fish community structure. In this paper we review the effects of electrofishing on fish and discuss this within the context of depletion sampling and multiple exposures of fishes to electric fields. The multiple wave forms most commonly used in sampling (alternating current, direct current, and pulsed direct current) are discussed as well as electrofishing induced response, injury and physiological stress. Fish that survive electrofishing injuries are more likely to suffer short and long-term adverse effects to their behavior, health, growth, or reproduction. Of greatest concern are the native, non-target species that may be subjected to multiple electrical shocks during the course of a 3-pass depletion survey. These exposures and their effects on the non-target species warrant further study as do the overall effects of electrofishing on populations and community structure. 

  12. [Authorization of pathologists for the estimation of the tumor cell percentage on tissue sample for molecular analysis purpose].

    PubMed

    Luquain, Alexandra; Arbez-Gindre, Francine; Bedgedjian, Isabelle; Felix, Sophie; Harimenshi, Jean-Marie; Mihai, Ionela-Marcela; Monnien, Franck; Singeorzan, Cristina; Valmary-Degano, Séverine

    2016-08-01

    Before molecular analysis is performed, morphological control with an estimation of the tumour cell percentage (%TC) could have a major impact on mutation detection. Accreditation according to NF EN ISO 15189 commands an authorization through evaluation of skills. The objective of this work was to validate the empowerment of pathologists to estimate %TC in tissue sample prior to molecular analysis. The accreditation technical guidance methods in Medical biology and histopathology were taken as references. %TC was the ratio of tumour cell nuclei on all nuclei within the area selected for the DNA extraction. External evaluations quality scores were used for accuracy. In order to assess the intermediate precision, 35 %TC estimation were performed 15 days apart in 4 samples (biopsies, transparietal biopsies or surgical specimen, either fixed or frozen) by 7 pathologists. Three other cases with interference (inflammation, mucus, necrosis) were evaluated. A result was satisfactory if %TC were within ±20 % of expected percentage obtained by the average of 35 estimates. The performances were satisfactory since no estimate was made more than 20 % of the expected percentage. Low interpathologists reproducibility has been reported in the literature and can have a consequence on molecular analysis in samples with low %TC, where the value reach the analytical sensitivity thresholds of molecular techniques. The current report is an example of a step of the accreditation process, which is a challenge for pathologists' activity in the future.

  13. Detection and quantification of Flavobacterium psychrophilum in water and fish tissue samples by quantitative real time PCR

    PubMed Central

    2014-01-01

    Background Flavobacterium psychrophilum is the agent of Bacterial Cold Water Disease and Rainbow Trout Fry Syndrome, two diseases leading to high mortality. Pathogen detection is mainly carried out using cultures and more rapid and sensitive methods are needed. Results We describe a qPCR technique based on the single copy gene β’ DNA-dependent RNA polymerase (rpoC). Its detection limit was 20 gene copies and the quantification limit 103 gene copies per reaction. Tests on spiked spleens with known concentrations of F. psychrophilum (106 to 101 cells per reaction) showed no cross-reactions between the spleen tissue and the primers and probe. Screening of water samples and spleens from symptomless and infected fishes indicated that the pathogen was already present before the outbreaks, but F. psychrophilum was only quantifiable in spleens from diseased fishes. Conclusions This qPCR can be used as a highly sensitive and specific method to detect F. psychrophilum in different sample types without the need for culturing. qPCR allows a reliable detection and quantification of F. psychrophilum in samples with low pathogen densities. Quantitative data on F. psychrophilum abundance could be useful to investigate risk factors linked to infections and also as early warning system prior to potential devastating outbreak. PMID:24767577

  14. Microwave-assisted tissue processing for same-day EM-diagnosis of potential bioterrorism and clinical samples.

    PubMed

    Schroeder, Josef A; Gelderblom, Hans R; Hauroeder, Baerbel; Schmetz, Christel; Milios, Jim; Hofstaedter, Ferdinand

    2006-01-01

    The purpose of this study was to explore the turnaround times, section and image quality of a number of more "difficult" specimens destined for rapid diagnostic electron microscopy (EM) after microwave-assisted processing. The results were assessed and compared with those of conventionally processed samples. A variety of infectious agents, some with a potential for bioterrorism, and liver biopsies serving as an example for routine histopathology samples were studied. The samples represented virus-producing cell cultures (such as SARS-coronavirus, West Nile virus, Orthopox virus), bacteria suspensions (cultures of Escherichia coli and genetically knockout apathogenic Bacillus anthracis), suspensions of parasites (malaria Plasmodium falciparum, Leishmania major, Microsporidia cuniculi, Caenorhabditis elegans), and whole Drosophila melanogaster flies infected with microsporidia. Fresh liver samples and infected flies were fixed in Karnovsky-fixative by microwaving (20 min), all other samples were fixed in buffered glutaraldehyde or Karnovsky-fixative overnight or longer. Subsequently, all samples were divided to evaluate alternative processing protocols: one part of the sample was OsO4-postfixed, ethanol-dehydrated, Epon-infiltrated (overnight) in an automated tissue processor (LYNX, Leica), and polymerized at 60 degrees C for 48 h; in parallel the other part was microwave-assisted processed in the bench microwave device (REM, Milestone), including post-osmication and the resin block polymerization. The microwave-assisted processing protocol required at minimum 3 h 20 min: the respective epon resin blocks were uniformly polymerized allowing an easy sectioning of semi- and ultrathin sections. Sections collected on non-coated 200 mesh grids were stable in the electron beam and showed an excellent preservation of the ultrastructure and high contrast, thus allowing an easy, unequivocal and rapid assessment of specimens. Compared with conventional routine methods

  15. Routine sample preparation and HPLC analysis for ascorbic acid (vitamin C) determination in wheat plants and Arabidopsis leaf tissues.

    PubMed

    Szalai, Gabriella; Janda, T; Pál, Magda

    2014-06-01

    Plants have developed various mechanisms to protect themselves against oxidative stress. One of the most important non-enzymatic antioxidants is ascorbic acid. There is thus a need for a rapid, sensitive method for the analysis of the reduced and oxidised forms of ascorbic acid in crop plants. In this paper a simple, economic, selective, precise and stable HPLC method is presented for the detection of ascorbate in plant tissue. The sensitivity, the short retention time and the simple isocratic elution mean that the method is suitable for the routine quantification of ascorbate in a high daily sample number. The method has been found to be better than previously reported methods, because of the use of an economical, readily available mobile phase, UV detection and the lack of complicated extraction procedures. The method has been tested on Arabidopsis plants with different ascorbate levels and on wheat plants during Cd stress.

  16. Gas-liquid chromatographic and gas-liquid-mass spectometric determination of fenvalerate and permethrin residues in grasshoppers and duck tissue samples

    USGS Publications Warehouse

    Reichel, W.L.; Kolbe, E.J.; Stafford, C.J.

    1981-01-01

    A procedure is described for determining fenvalerate and permethrin residues in grasshoppers and duck tissues. Samples are Soxhlet-extracted with hexane and cleaned up by gel permeation chromatography with an in-line alumina column. Samples are analyzed by gas-liquid chromatography with electron capture detection, and confirmed by gas-liquid chromatography-mass spectrometry. The average recovery from fortified tissues was 97%.

  17. Liquid microjunction surface sampling coupled with high-pressure liquid chromatography-electrospray ionization-mass spectrometry for analysis of drugs and metabolites in whole-body thin tissue sections.

    PubMed

    Kertesz, Vilmos; Van Berkel, Gary J

    2010-07-15

    In this work, a commercially available autosampler was adapted to perform direct liquid microjunction (LMJ) surface sampling followed by a high-pressure liquid chromatography (HPLC) separation of the extract components and detection with electrospray ionization mass spectrometry (ESI-MS). To illustrate the utility of coupling a separation with this direct liquid extraction based surface sampling approach, four different organs (brain, lung, kidney, and liver) from whole-body thin tissue sections of propranolol dosed and control mice were examined. The parent drug was observed in the chromatograms of the surface sampling extracts from all the organs of the dosed mouse examined. In addition, two isomeric phase II metabolites of propranolol (an aliphatic and an aromatic hydroxypropranolol glucuronide) were observed in the chromatograms of the extracts from lung, kidney, and liver. Confirming the presence of one or the other or both of these glucuronides in the extract from the various organs was not possible without the separation. These drug and metabolite data obtained using the LMJ surface sampling/HPLC-MS method and the results achieved by analyzing similar samples by conventional extraction of the tissues and subsequent HPLC-MS analysis were consistent. The ability to directly and efficiently sample from thin tissue sections via a liquid extraction and then perform a subsequent liquid phase separation increases the utility of this liquid extraction surface sampling approach.

  18. Exploring the cellular and tissue uptake of nanomaterials in a range of biological samples using multimodal nonlinear optical microscopy

    NASA Astrophysics Data System (ADS)

    Johnston, Helinor J.; Mouras, Rabah; Brown, David M.; Elfick, Alistair; Stone, Vicki

    2015-12-01

    The uptake of nanomaterials (NMs) by cells is critical in determining their potential biological impact, whether beneficial or detrimental. Thus, investigation of NM internalization by cells is a common consideration in hazard and efficacy studies. There are currently a number of approaches that are routinely used to investigate NM-cell interactions, each of which have their own advantages and limitations. Ideally, imaging modalities used to investigate NM uptake by cells should not require the NM to be labelled (e.g. with fluorophores) to facilitate its detection. We present a multimodal imaging approach employing a combination of label-free microscopies that can be used to investigate NM-cell interactions. Coherent anti-Stokes Raman scattering microscopy was used in combination with either two-photon photoluminescence or four-wave mixing (FWM) to visualize the uptake of gold or titanium dioxide NMs respectively. Live and fixed cell imaging revealed that NMs were internalized by J774 macrophage and C3A hepatocyte cell lines (15-31 μg ml-1). Sprague Dawley rats were exposed to NMs (intratracheal instillation, 62 μg) and NMs were detected in blood and lung leucocytes, lung and liver tissue, demonstrating that NMs could translocate from the exposure site. Obtained data illustrate that multimodal nonlinear optical microscopy may help overcome current challenges in the assessment of NM cellular uptake and biodistribution. It is therefore a powerful tool that can be used to investigate unlabelled NM cellular and tissue uptake in three dimensions, requires minimal sample preparation, and is applicable to live and fixed cells.

  19. The Tendon-to-Bone Transition of the Rotator Cuff: A Preliminary Raman Spectroscopic Study Documenting the Gradual Mineralization Across the Insertion in Rat Tissue Samples

    PubMed Central

    WOPENKA, BRIGITTE; KENT, ALISTAIR; PASTERIS, JILL D.; YOON, YOUNG; THOMOPOULOS, STAVROS

    2009-01-01

    We applied Raman spectroscopy to monitor the distribution of minerals and the degree of mineralization across the tendon–bone insertion site in the shoulders of five rats. We acquired Raman spectra from 100 to 4000 Δcm-1 on individual 1 μm points across the 120 μm wide transition zone of each tissue sample and identified all the peaks detected in pure tendon and in pure bone, as well as in the transition zone. The intensity of the 960 Δcm-1 P–O stretch for apatite (normalized to either the 2940 Δcm-1 C–H stretch or the 1003 Δcm-1 C–C stretch for collagen) was used as an indicator of the abundance of mineral. We relate the observed histological morphology in the tissue thin section with the observed Raman peaks for both the organic component (mostly collagen) and the inorganic component (a carbonated form of the mineral apatite) and discuss spectroscopic issues related to peak deconvolution and quantification of overlapping Raman peaks. We show that the mineral-to-collagen ratio at the insertion site increases linearly (R2 = 0.8 for five samples) over the distance of 120 μm from tendon to bone, rather than abruptly, as previously inferred from histological observations. In addition, narrowing of the 960 Δcm-1 band across the traverse indicates that the crystalline ordering within the apatite increases concomitantly with the degree of mineralization. This finding of mineral gradation has important clinical implications and may explain why the uninjured tendon-to-bone connection of the rotator cuff can sustain very high stress concentrations without failure. Our finding is also consistent with recent mechanical models and calculations developed to better understand the materials properties of this unusually strong interface. PMID:19094386

  20. A novel method for single sample multi-axial nanoindentation of hydrated heterogeneous tissues based on testing great white shark jaws.

    PubMed

    Ferrara, Toni L; Boughton, Philip; Slavich, Eve; Wroe, Stephen

    2013-01-01

    Nanomechanical testing methods that are suitable for a range of hydrated tissues are crucial for understanding biological systems. Nanoindentation of tissues can provide valuable insights into biology, tissue engineering and biomimetic design. However, testing hydrated biological samples still remains a significant challenge. Shark jaw cartilage is an ideal substrate for developing a method to test hydrated tissues because it is a unique heterogeneous composite of both mineralized (hard) and non-mineralized (soft) layers and possesses a jaw geometry that is challenging to test mechanically. The aim of this study is to develop a novel method for obtaining multidirectional nanomechanical properties for both layers of jaw cartilage from a single sample, taken from the great white shark (Carcharodon carcharias). A method for obtaining multidirectional data from a single sample is necessary for examining tissue mechanics in this shark because it is a protected species and hence samples may be difficult to obtain. Results show that this method maintains hydration of samples that would otherwise rapidly dehydrate. Our study is the first analysis of nanomechanical properties of great white shark jaw cartilage. Variation in nanomechanical properties were detected in different orthogonal directions for both layers of jaw cartilage in this species. The data further suggest that the mineralized layer of shark jaw cartilage is less stiff than previously posited. Our method allows multidirectional nanomechanical properties to be obtained from a single, small, hydrated heterogeneous sample. Our technique is therefore suitable for use when specimens are rare, valuable or limited in quantity, such as samples obtained from endangered species or pathological tissues. We also outline a method for tip-to-optic calibration that facilitates nanoindentation of soft biological tissues. Our technique may help address the critical need for a nanomechanical testing method that is applicable

  1. Percent recoveries of anthropogenic organic compounds with and without the addition of ascorbic acid to preserve finished-water samples containing free chlorine, 2004-10

    USGS Publications Warehouse

    Valder, Joshua F.; Delzer, Gregory C.; Bender, David A.; Price, Curtis V.

    2011-01-01

    This report presents finished-water matrix-spike recoveries of 270 anthropogenic organic compounds with and without the addition of ascorbic acid to preserve water samples containing free chlorine. Percent recoveries were calculated using analytical results from a study conducted during 2004-10 for the National Water-Quality Assessment (NAWQA) Program of the U.S. Geological Survey (USGS). The study was intended to characterize the effect of quenching on finished-water matrix-spike recoveries and to better understand the potential oxidation and transformation of 270 anthropogenic organic compounds. The anthropogenic organic compounds studied include those on analytical schedules 1433, 2003, 2033, 2060, 2020, and 4024 of the USGS National Water Quality Laboratory. Three types of samples were collected from 34 NAWQA locations across the Nation: (1) quenched finished-water samples (not spiked), (2) quenched finished-water matrix-spike samples, and (3) nonquenched finished-water matrix-spike samples. Percent recoveries of anthropogenic organic compounds in quenched and nonquenched finished-water matrix-spike samples are presented. Comparisons of percent recoveries between quenched and nonquenched spiked samples can be used to show how quenching affects finished-water samples. A maximum of 18 surface-water and 34 groundwater quenched finished-water matrix-spike samples paired with nonquenched finished-water matrix-spike samples were analyzed. Percent recoveries for the study are presented in two ways: (1) finished-water matrix-spike samples supplied by surface-water or groundwater, and (2) by use (or source) group category for surface-water and groundwater supplies. Graphical representations of percent recoveries for the quenched and nonquenched finished-water matrix-spike samples also are presented.

  2. Study Design and Percent Recoveries of Anthropogenic Organic Compounds With and Without the Addition of Ascorbic Acid to Preserve Water Samples Containing Free Chlorine, 2004-06

    USGS Publications Warehouse

    Valder, Joshua F.; Delzer, Gregory C.; Price, Curtis V.; Sandstrom, Mark W.

    2008-01-01

    The National Water-Quality Assessment (NAWQA) Program of the U.S. Geological Survey (USGS) began implementing Source Water-Quality Assessments (SWQAs) in 2002 that focus on characterizing the quality of source water and finished water of aquifers and major rivers used by some of the larger community water systems in the United States. As used for SWQA studies, source water is the raw (ambient) water collected at the supply well prior to water treatment (for ground water) or the raw (ambient) water collected from the river near the intake (for surface water). Finished water is the water that is treated, which typically involves, in part, the addition of chlorine or other disinfection chemicals to remove pathogens, and is ready to be delivered to consumers. Finished water is collected before the water enters the distribution system. This report describes the study design and percent recoveries of anthropogenic organic compounds (AOCs) with and without the addition of ascorbic acid to preserve water samples containing free chlorine. The percent recoveries were determined by using analytical results from a laboratory study conducted in 2004 by the USGS's National Water Quality Laboratory (NWQL) and from data collected during 2004-06 for a field study currently (2008) being conducted by the USGS's NAWQA Program. The laboratory study was designed to determine if preserving samples with ascorbic acid (quenching samples) adversely affects analytical performance under controlled conditions. During the laboratory study, eight samples of reagent water were spiked for each of five analytical schedules evaluated. Percent recoveries from these samples were then compared in two ways: (1) four quenched reagent spiked samples analyzed on day 0 were compared with four quenched reagent spiked samples analyzed on day 7 or 14, and (2) the combined eight quenched reagent spiked samples analyzed on day 0, 7, or 14 were compared with eight laboratory reagent spikes (LRSs). Percent

  3. Cell-type-specific expression of STAT transcription factors in tissue samples from patients with lymphocytic thyroiditis.

    PubMed

    Staab, Julia; Barth, Peter J; Meyer, Thomas

    2012-09-01

    Expression of cytokine-regulated signal transducer and activator of transcription (STAT) proteins was histochemically assessed in patients diagnosed as having Hashimoto's disease or focal lymphocytic thyroiditis (n = 10). All surgical specimens showed histological features of lymphocytic thyroiditis, including a diffuse infiltration with mononuclear cells and an incomplete loss of thyroid follicles, resulting in the destruction of glandular tissue architecture. Immunohistochemical analysis demonstrated differential expression patterns of the various members of the STAT transcription factors examined, indicating that each member of this conserved protein family has its distinct functions in the development of the disease. Using an antibody that specifically recognized the phosphorylated tyrosine residue in position 701, we detected activated STAT1 dimers in numerous germinal macrophages and infiltrating lymphocytes as well as in oncocytes. In contrast, STAT3 expression was restricted to epithelial cells and showed a clear colocalization with the antiapoptotic protein Bcl-2. Moreover, expression of phospho-STAT3 was associated with low levels of stromal fibrosis, suggesting that STAT3 serves as a protective factor in the remodeling of the inflamed thyroid gland. Phospho-STAT5 immunoreactivity was detected in numerous infiltrating cells of hematopoietic origin and, additionally, in hyperplastic follicular epithelia. This tissue distribution demonstrated that activated STAT5 molecules participate in both lymphocytopoiesis and possibly also in the buildup of regenerating thyroid follicles. Taken together, the cell-type-specific expression patterns of STAT proteins in human lymphocytic thyroiditis reflect their distinct and partially antagonistic roles in orchestrating the balance between degenerating and regenerating processes within a changing cytokine environment.

  4. Evaluation of an effective sample prefractionation method for the proteome analysis of breast cancer tissue using narrow range two-dimensional gel electrophoresis.

    PubMed

    Lee, KiBeom

    2008-06-01

    One method of improving the protein profiling of complex mammalian proteomes is the use of prefractionation followed by application of narrow pH range two dimensional (2-D) gels. The success of this strategy relies on sample solubilization; poor solubilization has been associated with missing protein fractions and diffuse, streaked, and/or trailing protein spots. In this study, I sought to optimize the solubilization of prefractionated human cancer cell samples using isoelectric focusing (IEF) rehydration buffers containing a variety of commercially available reducing agents, detergents, chaotropes, and carrier ampholytes. The solubilized proteins were resolved on 2-D gels and compared. Among five tested IEF rehydration buffers, those containing 3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS) and dithiothreitol (DTT) provided superior resolution, while that containing Nonidet P-40 (NP-40) did not significantly affect protein resolution, and the tributyl phosphine (TBP)-containing buffer yielded consistently poor results. In addition, I found that buffers containing typically high urea and ampholyte levels generated sharper 2-D gels. Using these optimized conditions, I was able to apply 2-D gel analysis successfully to fractionated proteins from human breast cancer tissue MCF-7, across a pH range of 4-6.7.

  5. The role of methanol addition to water samples in reducing analyte adsorption and matrix effects in liquid chromatography-tandem mass spectrometry.

    PubMed

    Li, Wei; Liu, Yucan; Duan, Jinming; Saint, Christopher P; Mulcahy, Dennis

    2015-04-10

    Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis coupled simply with water filtering before injection has proven to be a simple, economic and time-saving method for analyzing trace-level organic pollutants in aqueous environments. However, the linearity, precision and detection limits of such methods for late-eluting analytes were found to be much poorer than for early-eluting ones due to adsorption of the analytes in the operating system, such as sample vial, flow path and sample loop, creating problems in quantitative analysis. Addition of methanol (MeOH) into water samples as a modifier was shown to be effective in alleviating or even eliminating the negative effect on signal intensity for the late-eluting analytes and at the same time being able to reduce certain matrix effects for real water samples. Based on the maximum detection signal intensity obtained on desorption of the analytes with MeOH addition, the ratio of the detection signal intensity without addition of MeOH to the maximum intensity can be used to evaluate the effectiveness of methanol addition. Accordingly, the values of <50%, 50-80%, 80-120% could be used to indicate strong, medium and no effects, respectively. Based on this concept, an external matrix-matched calibration method with the addition of MeOH has been successfully established for analyzing fifteen pesticides with diverse physico-chemical properties in surface and groundwater with good linearity (r(2): 0.9929-0.9996), precision (intra-day relative standard deviation (RSD): 1.4-10.7%, inter-day RSD: 1.5-9.4%), accuracy (76.9-126.7%) and low limits of detection (0.003-0.028μg/L).

  6. Chemical compositions responsible for inflammation and tissue damage in the mouse lung by coarse and fine particulate samples from contrasting air pollution in Europe.

    PubMed

    Happo, Mikko S; Hirvonen, Maija-Riitta; Halinen, Arja I; Jalava, Pasi I; Pennanen, Arto S; Sillanpaa, Markus; Hillamo, Risto; Salonen, Raimo O

    2008-11-01

    Inflammation is regarded as an important mechanism in mortality and morbidity associated with exposures of cardiorespiratory patients to urban air particulate matter. We investigated the association of the chemical composition and sources of urban air fine (PM(2.5-0.2)) and coarse (PM(10-2.5)) particulate samples with the inflammatory activity in the mouse lung. The particulate samples were collected during selected seasons in six European cities using a high-volume cascade impactor. Healthy C57BL/6J mice were intratracheally instilled with a single dose (10 mg/kg) of the particulate samples. At 4, 12, and 24 h after the exposure, the lungs were lavaged and the bronchoalveolar lavage fluid (BALF) was assayed for indicators of inflammation and tissue damage: cell number, total protein, and cytokines (tumor necrosis factor [TNF]-alpha, interleukin [IL]-6, and KC). Dicarboxylic acids and transition metals, especially Ni and V, in PM(2.5-0.2) correlated positively and some secondary inorganic ions (NO3(-), NH4(+)) negatively with the inflammatory activity. Total organic matter and SO4(2-) had no consistent correlations. In addition, the soil-derived constituents (Ca2+, Al, Fe, Si) showed positive correlations with the PM(2.5-0.2)-induced inflammatory activity, but their role in PM(10-2.5) remained obscure, possibly due to largely undefined biogenic material. Markers of poor biomass and coal combustion, i.e., monosaccharide anhydrides and As, were associated with elevated PAH contents in PM(2.5-0.2) and a consistent immunosuppressive effect. Overall, our results support epidemiological findings that the local sources of incomplete combustion and resuspended road dust are important in urban air particulate pollution-related health effects.

  7. Heat treatment and the use of additives to improve the stability of paralytic shellfish poisoning toxins in shellfish tissue reference materials for internal quality control and proficiency testing.

    PubMed

    Burrell, Stephen; Clion, Valentin; Auroy, Virginie; Foley, Barry; Turner, Andrew D

    2015-06-01

    The need for homogenous reference materials stable for paralytic shellfish toxins is vital for the monitoring and quality assurance of these potent neurotoxins in shellfish. Two stabilisation techniques were investigated, heat treatment through autoclaving and the addition of preserving additives into the tissue matrix. Short and long-term stability experiments as well as homogeneity determination were conducted on materials prepared by both techniques in comparison with an untreated control using two LC-FLD methods. Both techniques improved the stability of the matrix and the PSP toxins present compared to the controls. A material was prepared using the combined techniques of heat treatment followed by spiking with additives and data is presented from this optimised reference material as used over a two year period in the Irish national monitoring program and in a development exercise as part of a proficiency testing scheme operated by QUASIMEME (Quality Assurance of Information for Marine Environmental Monitoring in Europe) since 2011. The results were indicative of the long-term stability of the material as evidenced through consistent assigned values in the case of the proficiency testing scheme and a low relative standard deviation of 10.5% for total toxicity data generated over 24 months.

  8. Preparation of an additive-free sample with a MgH2 phase by planetary ball milling of Mg with10 wt% MgH2

    NASA Astrophysics Data System (ADS)

    Hong, Seong-Hyeon; Song, Myoung Youp

    2016-11-01

    In order to prepare an additive-free sample with a MgH2 phase, 90 wt% Mg+10 wt% MgH2 (named Mg-10MgH2) was milled under hydrogen atmosphere in a planetary ball mill for different durations (2 h, 5 h, and 10 h). The hydrogen absorption and release properties of the prepared samples were investigated and compared with those of purchased pure MgH2 samples. Mg-10MgH2 milled for 5 h had the largest quantity of hydrogen released at 648 K for 100 min of 5.96 wt%. Mg-10MgH2 milled for 5 h released 0.11 wt% H for 10 min, 4.85 wt% H for 30 min, and 5.83 wt% H for 60 min at 648 K at the first cycle. Mg-10MgH2 milled for 5 h absorbed 5.39 wt% H for 5 min and 5.92 wt% H for 60 min at 648 K at the second cycle. Dehydriding curves were also obtained at the first cycle of Mg-10MgH2 samples milled for 5 h using Mg powder with or without sieving (200 mesh). The dehydriding curve at 648 K of a Mg-10MgH2 sample milled for 5 h in the planetary ball mill was compared with that of the sample milled for 24 h in a horizontal ball mill.

  9. Selective culturing and genus-specific PCR detection for identification of Aeromonas in tissue samples to assist the medico-legal diagnosis of death by drowning.

    PubMed

    Huys, Geert; Coopman, Vera; Van Varenbergh, Dirk; Cordonnier, Jan

    2012-09-10

    The detection of autochthonous aquatic bacteria in tissue samples from drowning cases is increasingly considered as an alternative approach to assist the medico-legal diagnosis of death by drowning. Bacteria belonging to the genus Aeromonas may be suitable candidates for this application as they are ubiquitous in natural aquatic environments but are generally not part of the human microbiota. The research aims of this study were (i) to develop a sensitive, specific and rapid screening and confirmation method for Aeromonas species in tissue samples and (ii) to evaluate aseptic sternal puncture as a post-mortem sample technique and bone marrow as an alternative matrix to provide evidence of death by drowning. The presence of Aeromonas in tissue samples was verified by cultivation using the selective media Ampicillin Dextrin Agar (ADA) and Ryan's Aeromonas Medium. The use of ADA medium was found most optimal for the sensitive, inexpensive and quick detection of aeromonads in human tissue samples. Positive culture plates were confirmed by harvesting all colonies for DNA extraction and subsequent PCR amplification using Aeromonas genus-specific primers. Aeromonads were detected in lung swab, blood and bone marrow of drowned bodies (n=3), but were negative in these three matrices for all negative controls (n=90) tested. Bone marrow proved to be a suitable alternative matrix and can be sampled post-mortem by an aseptic sternal puncture. In conclusion, this study confirms previous indications that aeromonads in cultures from blood of water bodies can be considered a potential marker for drowning. Given the fact that the number of immersed bodies (drowned and non-drowned) included in this study is statistically not significant, however, more tissue samples need to be investigated to confirm the validity of these methods to aid the diagnosis of death by wet drowning.

  10. Influence of sample preparation and reliability of automated numerical refocusing in stain-free analysis of dissected tissues with quantitative phase digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Lenz, Philipp; Bettenworth, Dominik; Krausewitz, Philipp; Domagk, Dirk; Ketelhut, Steffi

    2015-05-01

    Digital holographic microscopy (DHM) has been demonstrated to be a versatile tool for high resolution non-destructive quantitative phase imaging of surfaces and multi-modal minimally-invasive monitoring of living cell cultures in-vitro. DHM provides quantitative monitoring of physiological processes through functional imaging and structural analysis which, for example, gives new insight into signalling of cellular water permeability and cell morphology changes due to toxins and infections. Also the analysis of dissected tissues quantitative DHM phase contrast prospects application fields by stain-free imaging and the quantification of tissue density changes. We show that DHM allows imaging of different tissue layers with high contrast in unstained tissue sections. As the investigation of fixed samples represents a very important application field in pathology, we also analyzed the influence of the sample preparation. The retrieved data demonstrate that the quality of quantitative DHM phase images of dissected tissues depends strongly on the fixing method and common staining agents. As in DHM the reconstruction is performed numerically, multi-focus imaging is achieved from a single digital hologram. Thus, we evaluated the automated refocussing feature of DHM for application on different types of dissected tissues and revealed that on moderately stained samples highly reproducible holographic autofocussing can be achieved. Finally, it is demonstrated that alterations of the spatial refractive index distribution in murine and human tissue samples represent a reliable absolute parameter that is related of different degrees of inflammation in experimental colitis and Crohn's disease. This paves the way towards the usage of DHM in digital pathology for automated histological examinations and further studies to elucidate the translational potential of quantitative phase microscopy for the clinical management of patients, e.g., with inflammatory bowel disease.

  11. Evaluation of cytokeratin-19 in breast cancer tissue samples: a comparison of automatic and manual evaluations of scanned tissue microarray cylinders

    PubMed Central

    2015-01-01

    Background Digital image (DI) analysis avoids visual subjectivity in interpreting immunohistochemical stains and provides more reproducible results. An automated procedure consisting of two variant methods for quantifying the cytokeratin-19 (CK19) marker in breast cancer tissues is presented. Methods The first method (A) excludes the holes inside selected CK19 stained areas, and the second (B) includes them. 93 DIs scanned from complete cylinders of tissue microarrays were evaluated visually by two pathologists and by the automated procedures. Results and conclusions There was good concordance between the two automated methods, both of which tended to identify a smaller CK19-positive area than did the pathologists. The results obtained with method B were more similar to those of the pathologists; probably because it takes into account the entire positive tumoural area, including the holes. However, the pathologists overestimated the positive area of CK19. Further studies are needed to confirm the utility of this automated procedure in prognostic studies. PMID:26329009

  12. Comparison of Nanostring nCounter® Data on FFPE Colon Cancer Samples and Affymetrix Microarray Data on Matched Frozen Tissues.

    PubMed

    Chen, Xi; Deane, Natasha G; Lewis, Keeli B; Li, Jiang; Zhu, Jing; Washington, M Kay; Beauchamp, R Daniel

    2016-01-01

    The prognosis of colorectal cancer (CRC) stage II and III patients remains a challenge due to the difficulties of finding robust biomarkers suitable for testing clinical samples. The majority of published gene signatures of CRC have been generated on fresh frozen colorectal tissues. Because collection of frozen tissue is not practical for routine surgical pathology practice, a clinical test that improves prognostic capabilities beyond standard pathological staging of colon cancer will need to be designed for formalin-fixed paraffin-embedded (FFPE) tissues. The NanoString nCounter® platform is a gene expression analysis tool developed for use with FFPE-derived samples. We designed a custom nCounter® codeset based on elements from multiple published fresh frozen tissue microarray-based prognostic gene signatures for colon cancer, and we used this platform to systematically compare gene expression data from FFPE with matched microarray array data from frozen tissues. Our results show moderate correlation of gene expression between two platforms and discovery of a small subset of genes as candidate biomarkers for colon cancer prognosis that are detectable and quantifiable in FFPE tissue sections.

  13. A Comparison of RNA-Seq Results from Paired Formalin-Fixed Paraffin-Embedded and Fresh-Frozen Glioblastoma Tissue Samples

    PubMed Central

    Esteve-Codina, Anna; Arpi, Oriol; Martinez-García, Maria; Pineda, Estela; Mallo, Mar; Gut, Marta; Carrato, Cristina; Rovira, Anna; Lopez, Raquel; Tortosa, Avelina; Dabad, Marc; Del Barco, Sonia; Heath, Simon; Bagué, Silvia; Ribalta, Teresa; Alameda, Francesc; de la Iglesia, Nuria

    2017-01-01

    The molecular classification of glioblastoma (GBM) based on gene expression might better explain outcome and response to treatment than clinical factors. Whole transcriptome sequencing using next-generation sequencing platforms is rapidly becoming accepted as a tool for measuring gene expression for both research and clinical use. Fresh frozen (FF) tissue specimens of GBM are difficult to obtain since tumor tissue obtained at surgery is often scarce and necrotic and diagnosis is prioritized over freezing. After diagnosis, leftover tissue is usually stored as formalin-fixed paraffin-embedded (FFPE) tissue. However, RNA from FFPE tissues is usually degraded, which could hamper gene expression analysis. We compared RNA-Seq data obtained from matched pairs of FF and FFPE GBM specimens. Only three FFPE out of eleven FFPE-FF matched samples yielded informative results. Several quality-control measurements showed that RNA from FFPE samples was highly degraded but maintained transcriptomic similarities to RNA from FF samples. Certain issues regarding mutation analysis and subtype prediction were detected. Nevertheless, our results suggest that RNA-Seq of FFPE GBM specimens provides reliable gene expression data that can be used in molecular studies of GBM if the RNA is sufficiently preserved. PMID:28122052

  14. High-resolution MALDI-FT-ICR MS imaging for the analysis of metabolites from formalin-fixed, paraffin-embedded clinical tissue samples.

    PubMed

    Buck, Achim; Ly, Alice; Balluff, Benjamin; Sun, Na; Gorzolka, Karin; Feuchtinger, Annette; Janssen, Klaus-Peter; Kuppen, Peter J K; van de Velde, Cornelis J H; Weirich, Gregor; Erlmeier, Franziska; Langer, Rupert; Aubele, Michaela; Zitzelsberger, Horst; Aichler, Michaela; Walch, Axel

    2015-09-01

    We present the first analytical approach to demonstrate the in situ imaging of metabolites from formalin-fixed, paraffin-embedded (FFPE) human tissue samples. Using high-resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR MSI), we conducted a proof-of-principle experiment comparing metabolite measurements from FFPE and fresh frozen tissue sections, and found an overlap of 72% amongst 1700 m/z species. In particular, we observed conservation of biomedically relevant information at the metabolite level in FFPE tissues. In biomedical applications, we analysed tissues from 350 different cancer patients and were able to discriminate between normal and tumour tissues, and different tumours from the same organ, and found an independent prognostic factor for patient survival. This study demonstrates the ability to measure metabolites in FFPE tissues using MALDI-FT-ICR MSI, which can then be assigned to histology and clinical parameters. Our approach is a major technical, histochemical, and clinicopathological advance that highlights the potential for investigating diseases in archived FFPE tissues.

  15. Analytical procedure for mapping the distribution of 10B and 99Tc markers in cryo-sections of animal tissue samples by secondary ion mass spectrometry

    NASA Astrophysics Data System (ADS)

    Marchetti, Ilaria; Menichetti, Luca; Kusmic, Claudia; de las Heras, Laura Aldave; Salvadori, Piero; Fuoco, Roger; Belloni, Fabio; L'Abbate, Antonio; Betti, Maria

    2009-09-01

    The development of a complete, standard analytical procedure for a quantitative use of secondary ion mass spectrometry to map the distribution in animal tissues of exogenous isotopes presents difficulties inherently related to sample preparation and preservation, as well as to the specific application being considered. We have tested in two very different cases a procedure based on the cryo-preparation of samples and calibration standards. The applications under investigation were the mapping of 10B in mouse brain tissue, with relevance to the boron neutron capture therapy, and of the perfusion tracer 99Tc in mouse heart tissue, with relevance to the study of microcirculation and cardiovascular pathologies. Scanning electron microscopy and inductively coupled mass spectrometry analysis were used as reference techniques for secondary ion mass spectrometry images and analyte measurements, respectively. Cryo-preparation of tissue sections for ion microscopy proved to be simple and efficient (in terms of structural and chemical integrity) for both brain and heart samples derived from fresh organs. This technique, however, turned out to be reliable only on the brain tissue when applied to the preparation of standards, which required chemical fixation of portions of organs. Brain and heart tissues showed a totally different response to chemical fixation, from both a structural and an analytical point of view. On the one hand, we were able to estimate a relative sensitivity factor for 10B in the cryo-sectioned brain matrix; on the other hand, even without the possibility of an absolute quantification of the 99Tc signal and notwithstanding the presence of an isobaric interference, secondary ion mass spectrometry mapping however proved to be capable to resolve the specific response of the cardiac tissue to the perfusion mechanism.

  16. Stochastic sampled-data control for synchronization of complex dynamical networks with control packet loss and additive time-varying delays.

    PubMed

    Rakkiyappan, R; Sakthivel, N; Cao, Jinde

    2015-06-01

    This study examines the exponential synchronization of complex dynamical networks with control packet loss and additive time-varying delays. Additionally, sampled-data controller with time-varying sampling period is considered and is assumed to switch between m different values in a random way with given probability. Then, a novel Lyapunov-Krasovskii functional (LKF) with triple integral terms is constructed and by using Jensen's inequality and reciprocally convex approach, sufficient conditions under which the dynamical network is exponentially mean-square stable are derived. When applying Jensen's inequality to partition double integral terms in the derivation of linear matrix inequality (LMI) conditions, a new kind of linear combination of positive functions weighted by the inverses of squared convex parameters appears. In order to handle such a combination, an effective method is introduced by extending the lower bound lemma. To design the sampled-data controller, the synchronization error system is represented as a switched system. Based on the derived LMI conditions and average dwell-time method, sufficient conditions for the synchronization of switched error system are derived in terms of LMIs. Finally, numerical example is employed to show the effectiveness of the proposed methods.

  17. A new method for the treatment of sperm samples for ultrastructural study based on the use of animal tissues as biological containers.

    PubMed

    Junquera, Concepción; Colás, Carmen; Martínez-Ciriano, Carmen; Serrano, Pedro; Castiella, Tomás; Cebrian-Perez, José Alvaro; Muiño-Blanco, Teresa

    2007-09-01

    The study of the ultrastructure of spematozoa by means of transmission electron microscopy often presents with problems of interpretation according to the method employed, depending on whether samples are either centrifuged previously to the fixation or immersed in viscous gels. The major problems of interpretation are: changes in the location of vesicles originated during the maturation process and modifications in the adsorption of seminal plasma proteins to the sperm membrane surface. The aim of our study is to communicate an original new method for the treatment of spermatozoa for ultrastructural study. Our method is based on the use of animal tissues as biological containers, inside which the spermatic suspensions are included. We developed this method using fresh sperm samples taken from mature Rasa aragonesa rams. As biological container, we used 2.5-cm long segments of the intestine of 1-week-old chickens (Gallus gallus) (diameter around 4 mm). To avoid any influence of digestive enzymes of the mucosa on the sperm surface, we put each intestine fragment inside out by means of microdissection forceps under bifocal optical microscope and cold light. One of the edges was tied with thin suture silk. The sperm suspension was injected in the optimal experimental condition and amount. Finally, the still open edge of the intestine segment was tied with silk in the same way as the other segment edge. By using this technique, we can perform a suitable morphological study at an ultrastructural level. In addition, the functional relationship of the ultrastructural components of the target cells is correctly preserved.

  18. New method for the treatment of sperm samples for ultrastructural study based on the use of animal tissues as biological containers.

    PubMed

    Junquera, Concepción; Colás, Carmen; Martínez-Ciriano, Carmen; Serrano, Pedro; Castiella, Tomás; Cebrian-Perez, José A; Muiño-Blanco, Teresa

    2007-08-01

    The study of the ultrastructure of spematozoa by means of transmission electron microscopy (TEM) often presents with problems of interpretation according to the method employed, depending on whether samples are either centrifuged previously to the fixation or immersed in viscous gels. The major problems of interpretation are changes in the location of vesicles originated during the maturation process and modifications in the adsorption of seminal plasma proteins to the sperm membrane surface. The aim of our study is to communicate an original new method for the treatment of spermatozoa for ultrastructural study. Our method is based on the use of animal tissues as biological containers, inside which the spermatic suspensions are included. We developed this method using fresh sperm samples taken from mature Rasa Aragonesa rams. As biological container, we used 2.5-cm long segments of the intestine of 1-week-old chickens (Gallus gallus) (diameter around 4 mm). In order to avoid any influence of digestive enzymes of the mucosa on the sperm surface, we put each intestine fragment inside out by means of microdissection forceps under a bifocal optical microscope and cold light. One of the edges was tied with thin suture silk. The sperm suspension was injected in the optimal experimental condition and amount. Finally, the still-open edge of the intestine segment was tied with silk in the same way as the other segment edge. By using this technique, we can perform a suitable morphological study at an ultrastructural level. In addition, the functional relationship of the ultrastructural components of the target cells is correctly preserved.

  19. A Core Invasiveness Gene Signature Reflects Epithelial-to-Mesenchymal Transition but Not Metastatic Potential in Breast Cancer Cell Lines and Tissue Samples

    PubMed Central

    Marsan, Melike; Van den Eynden, Gert; Limame, Ridha; Neven, Patrick; Hauspy, Jan; Van Dam, Peter A.; Vergote, Ignace; Dirix, Luc Y.; Vermeulen, Peter B.; Van Laere, Steven J.

    2014-01-01

    Introduction Metastases remain the primary cause of cancer-related death. The acquisition of invasive tumour cell behaviour is thought to be a cornerstone of the metastatic cascade. Therefore, gene signatures related to invasiveness could aid in stratifying patients according to their prognostic profile. In the present study we aimed at identifying an invasiveness gene signature and investigated its biological relevance in breast cancer. Methods & Results We collected a set of published gene signatures related to cell motility and invasion. Using this collection, we identified 16 genes that were represented at a higher frequency than observed by coincidence, hereafter named the core invasiveness gene signature. Principal component analysis showed that these overrepresented genes were able to segregate invasive and non-invasive breast cancer cell lines, outperforming sets of 16 randomly selected genes (all P<0.001). When applied onto additional data sets, the expression of the core invasiveness gene signature was significantly elevated in cell lines forced to undergo epithelial-mesenchymal transition. The link between core invasiveness gene expression and epithelial-mesenchymal transition was also confirmed in a dataset consisting of 2420 human breast cancer samples. Univariate and multivariate Cox regression analysis demonstrated that CIG expression is not associated with a shorter distant metastasis free survival interval (HR = 0.956, 95%C.I. = 0.896–1.019, P = 0.186). Discussion These data demonstrate that we have identified a set of core invasiveness genes, the expression of which is associated with epithelial-mesenchymal transition in breast cancer cell lines and in human tissue samples. Despite the connection between epithelial-mesenchymal transition and invasive tumour cell behaviour, we were unable to demonstrate a link between the core invasiveness gene signature and enhanced metastatic potential. PMID:24586640

  20. Additional effect of metformin and celecoxib against lipid dysregulation and adipose tissue inflammation in high-fat fed rats with insulin resistance and fatty liver.

    PubMed

    Lu, Chieh-Hua; Hung, Yi-Jen; Hsieh, Po-Shiuan

    2016-10-15

    We investigated the effects of metformin and celecoxib on obesity-induced adipose tissue inflammation, insulin resistance (IR), fatty liver, and high blood pressure in high-fat (HF) fed rats. Male Sprague-Dawley rats were fed with either regular or HF diet for 8 weeks. Rats fed with regular diet were treated with vehicle for further 4 weeks. HF fed rats were divided into 6 groups, namely, vehicle, celecoxib (30mg/kg/day), metformin (300mg/kg/day), metformin (150mg/kg/day), metformin (300mg/kg/day) with celecoxib (30mg/kg/day), and metformin (150mg/kg/day) with celecoxib (15mg/kg/day) for additional 4 weeks. Increased body weight in HF fed rats was significantly reduced by metformin alone and metformin combined with celecoxib. The increases in the HOMA-IR value and the area under the curve of glucose following an oral glucose tolerance test, systolic blood pressure, and adipocyte size were significantly diminished in treated rats, especially rats undergoing combined treatment. Treatments with either celecoxib or in combination with metformin resulted in a reduction in AT macrophage infiltration and decreases in levels of adipose tissue TNF-α, MCP-1, and leptin levels in high-fat (HF) fed rats. Furthermore, the elevated hepatic triglycerides content was significantly decreased in the combined treatment group compared to that of groups of celecoxib or metformin alone. Celecoxib exerts a synergistic beneficial effect with metformin on and obesity-associated metabolic and cardiovascular disorders in high-fat fed rats.

  1. Color Tissue Doppler to Analyze Fetal Cardiac Time Intervals: Normal Values and Influence of Sample Gate Size.

    PubMed

    Willruth, A M; Steinhard, J; Enzensberger, C; Axt-Fliedner, R; Gembruch, U; Doelle, A; Dimitriou, I; Fimmers, R; Bahlmann, F

    2016-02-04

    Purpose: To assess the time intervals of the cardiac cycle in healthy fetuses in the second and third trimester using color tissue Doppler imaging (cTDI) and to evaluate the influence of different sizes of sample gates on time interval values. Materials and Methods: Time intervals were measured from the cTDI-derived Doppler waveform using a small and large region of interest (ROI) in healthy fetuses. Results: 40 fetuses were included. The median gestational age at examination was 26 + 1 (range: 20 + 5 - 34 + 5) weeks. The median frame rate was 116/s (100 - 161/s) and the median heart rate 143 (range: 125 - 158) beats per minute (bpm). Using small and large ROIs, the second trimester right ventricular (RV) mean isovolumetric contraction times (ICTs) were 39.8 and 41.4 ms (p = 0.17), the mean ejection times (ETs) were 170.2 and 164.6 ms (p < 0.001), the mean isovolumetric relaxation times (IRTs) were 52.8 and 55.3 ms (p = 0.08), respectively. The left ventricular (LV) mean ICTs were 36.2 and 39.4 ms (p = 0.05), the mean ETs were 167.4 and 164.5 ms (p = 0.013), the mean IRTs were 53.9 and 57.1 ms (p = 0.05), respectively. The third trimester RV mean ICTs were 50.7 and 50.4 ms (p = 0.75), the mean ETs were 172.3 and 181.4 ms (p = 0.49), the mean IRTs were 50.2 and 54.6 ms (p = 0.03); the LV mean ICTs were 45.1 and 46.2 ms (p = 0.35), the mean ETs were 175.2 vs. 172.9 ms (p = 0.29), the mean IRTs were 47.1 and 50.0 ms (p = 0.01), respectively. Conclusion: Isovolumetric time intervals can be analyzed precisely and relatively independent of ROI size. In the near future, automatic time interval measurement using ultrasound systems will be feasible and the analysis of fetal myocardial function can become part of the clinical routine.

  2. Profiling of adrenocorticotropic hormone and arginine vasopressin in human pituitary gland and tumor thin tissue sections using droplet-based liquid-microjunction surface-sampling-HPLC-ESI-MS-MS.

    PubMed

    Kertesz, Vilmos; Calligaris, David; Feldman, Daniel R; Changelian, Armen; Laws, Edward R; Santagata, Sandro; Agar, Nathalie Y R; Van Berkel, Gary J

    2015-08-01

    Described here are the results from the profiling of the proteins arginine vasopressin (AVP) and adrenocorticotropic hormone (ACTH) from normal human pituitary gland and pituitary adenoma tissue sections, using a fully automated droplet-based liquid-microjunction surface-sampling-HPLC-ESI-MS-MS system for spatially resolved sampling, HPLC separation, and mass spectrometric detection. Excellent correlation was found between the protein distribution data obtained with this method and data obtained with matrix-assisted laser desorption/ionization (MALDI) chemical imaging analyses of serial sections of the same tissue. The protein distributions correlated with the visible anatomic pattern of the pituitary gland. AVP was most abundant in the posterior pituitary gland region (neurohypophysis), and ATCH was dominant in the anterior pituitary gland region (adenohypophysis). The relative amounts of AVP and ACTH sampled from a series of ACTH-secreting and non-secreting pituitary adenomas correlated with histopathological evaluation. ACTH was readily detected at significantly higher levels in regions of ACTH-secreting adenomas and in normal anterior adenohypophysis compared with non-secreting adenoma and neurohypophysis. AVP was mostly detected in normal neurohypophysis, as expected. This work reveals that a fully automated droplet-based liquid-microjunction surface-sampling system coupled to HPLC-ESI-MS-MS can be readily used for spatially resolved sampling, separation, detection, and semi-quantitation of physiologically-relevant peptide and protein hormones, including AVP and ACTH, directly from human tissue. In addition, the relative simplicity, rapidity, and specificity of this method support the potential of this basic technology, with further advancement, for assisting surgical decision-making. Graphical Abstract Mass spectrometry based profiling of hormones in human pituitary gland and tumor thin tissue sections.

  3. Influence of parasite density and sample storage time on the reliability of Entamoeba histolytica-specific PCR from formalin-fixed and paraffin-embedded tissues.

    PubMed

    Frickmann, Hagen; Tenner-Racz, Klara; Eggert, Petra; Schwarz, Norbert G; Poppert, Sven; Tannich, Egbert; Hagen, Ralf M

    2013-12-01

    We report on the reliability of polymerase chain reaction (PCR) for the detection of Entamoeba histolytica from formalin-fixed, paraffin-embedded tissue in comparison with microscopy and have determined predictors that may influence PCR results. E. histolytica-specific and Entamoeba dispar-specific real-time PCR and microscopy from adjacent histologic sections were performed using a collection of formalin-fixed, paraffin-embedded tissue specimens obtained from patients with invasive amebiasis. Specimens had been collected during the previous 4 decades. Association of sample age, parasite density, and reliability of PCR was analyzed. E. histolytica PCR was positive in 20 of 34 biopsies (58.8%); 2 of these 20 were microscopically negative for amebae in neighboring tissue sections. PCR was negative in 9 samples with visible amebae in neighboring sections and in 5 samples without visible parasites in neighboring sections. PCR was negative in all specimens that were older than 3 decades. Low parasite counts and sample ages older than 20 years were predictors for false-negative PCR results. All samples were negative for E. dispar DNA. PCR is suitable for the detection of E. histolytica in formalin-fixed, paraffin-embedded tissue samples that are younger than 2 decades and that contain intermediate to high parasite numbers. Negative results in older samples were due to progressive degradation of DNA over time as indicated by control PCRs targeting the human 18S rRNA gene. Moreover, our findings support previous suggestions that only E. histolytica but not E. dispar is responsible for invasive amebiasis.

  4. Synchrotron-based FTIR microspectroscopy for the mapping of photo-oxidation and additives in acrylonitrile-butadiene-styrene model samples and historical objects.

    PubMed

    Saviello, Daniela; Pouyet, Emeline; Toniolo, Lucia; Cotte, Marine; Nevin, Austin

    2014-09-16

    Synchrotron-based Fourier transform infrared micro-spectroscopy (SR-μFTIR) was used to map photo-oxidative degradation of acrylonitrile-butadiene-styrene (ABS) and to investigate the presence and the migration of additives in historical samples from important Italian design objects. High resolution (3×3 μm(2)) molecular maps were obtained by FTIR microspectroscopy in transmission mode, using a new method for the preparation of polymer thin sections. The depth of photo-oxidation in samples was evaluated and accompanied by the formation of ketones, aldehydes, esters, and unsaturated carbonyl compounds. This study demonstrates selective surface oxidation and a probable passivation of material against further degradation. In polymer fragments from design objects made of ABS from the 1960s, UV-stabilizers were detected and mapped, and microscopic inclusions of proteinaceous material were identified and mapped for the first time.

  5. Some Physical, Chemical, and Biological Parameters of Samples of Scleractinium Coral Aquaculture Skeleton Used for Reconstruction/Engineering of the Bone Tissue.

    PubMed

    Popov, A A; Sergeeva, N S; Britaev, T A; Komlev, V S; Sviridova, I K; Kirsanova, V A; Akhmedova, S A; Dgebuadze, P Yu; Teterina, A Yu; Kuvshinova, E A; Schanskii, Ya D

    2015-08-01

    Physical and chemical (phase and chemical composition, dynamics of resorption, and strength properties), and biological (cytological compatibility and scaffold properties of the surface) properties of samples of scleractinium coral skeletons from aquacultures of three types and corresponding samples of natural coral skeletons (Pocillopora verrucosa, Acropora formosa, and Acropora nobilis) were studied. Samples of scleractinium coral aquaculture skeleton of A. nobilis, A. formosa, and P. verrucosa met the requirements (all study parameters) to materials for osteoplasty and 3D-scaffolds for engineering of bone tissue.

  6. QA/QC Guidance for Sampling and Analysis of Sediments, Water, and Tissues for Dredged Material Evaluations: Chemical Evaluations

    DTIC Science & Technology

    1995-04-01

    0; 6010A/200.7 Recommended Water TDLb 40 3 1 I 0.2 1 I 1 TABLE 3. (cont.) Chemical Cobalt Copper Hexavalent chromium Iron Lead...type of sample (e.g., C =sediment for chemistry and bioassay analyses, B = bioaccumulation, or I = benthic infauna). • The resulting sample...identifier would be: BHS01 C. When field replicates are collected (i.e., for benthic samples), the replicate number should be appended to the sample

  7. The effect of co-occurring polychlorinated biphenyls on quantitation of toxaphene in fish tissue samples by gas chromatography negative ion mass spectrometry.

    PubMed

    Lao, Wenjian; Tsukada, David; Maruya, Keith A

    2012-12-28

    Determinative methods based on gas chromatography-negative chemical ionization mass spectrometry (GC-NCI/MS) provide improved sensitivity and specificity for toxaphene in environmental samples, but are subject to misidentification due to oxygen reaction in the presence of polychlorinated biphenyls (PCBs). The goal of this study was to quantify the impact of co-occurring PCBs in fish tissue samples when utilizing single quadrupole instruments to implement this method. Mixtures of PCB congeners and technical toxaphene, and extracts of fish tissue with varying concentrations of PCBs were analyzed for individual congener and total toxaphene concentrations by GC-NCI/MS. The contribution of co-injected PCB 204 ranged from 23% to 88% of the total peak area for the Cl-9 toxaphene homolog quantitation ion, a contribution that increased as the ratio of technical toxaphene to PCB 204 decreased. PCB interferences in fish tissue extracts, including a standard reference material, were subtracted using a three-step procedure featuring spectral analysis of isotopic patterns for target peaks. Total toxaphene concentrations without PCB subtraction in three fish tissue samples with low, intermediate and high co-occurring PCBs were overestimated by 33, 55 and 745%, respectively, underscoring the need for practical strategies to account for PCB interferences in GC-NCI/MS based protocols. In contrast, no appreciable interference or resulting positive bias in concentrations was observed for quantitation of eight common toxaphene residue congeners.

  8. Profiling of adrenocorticotropic hormone and arginine vasopressin in human pituitary gland and tumor thin tissue sections using droplet-based liquid-microjunction surface-sampling-HPLC–ESI-MS–MS

    DOE PAGES

    Kertesz, Vilmos; Calligaris, David; Feldman, Daniel R.; ...

    2015-06-18

    Described here are the results from the profiling of the proteins arginine vasopressin (AVP) and adrenocorticotropic hormone (ACTH) from normal human pituitary gland and pituitary adenoma tissue sections using a fully automated droplet-based liquid microjunction surface sampling-HPLC-ESI-MS/MS system for spatially resolved sampling, HPLC separation, and mass spectral detection. Excellent correlation was found between the protein distribution data obtained with this droplet-based liquid microjunction surface sampling-HPLC-ESI-MS/MS system and those data obtained with matrix assisted laser desorption ionization (MALDI) chemical imaging analyses of serial sections of the same tissue. The protein distributions correlated with the visible anatomic pattern of the pituitary gland.more » AVP was most abundant in the posterior pituitary gland region (neurohypophysis) and ATCH was dominant in the anterior pituitary gland region (adenohypophysis). The relative amounts of AVP and ACTH sampled from a series of ACTH secreting and non-secreting pituitary adenomas correlated with histopathological evaluation. ACTH was readily detected at significantly higher levels in regions of ACTH secreting adenomas and in normal anterior adenohypophysis compared to non-secreting adenoma and neurohypophysis. AVP was mostly detected in normal neurohypophysis as anticipated. This work demonstrates that a fully automated droplet-based liquid microjunction surface sampling system coupled to HPLC-ESI-MS/MS can be readily used for spatially resolved sampling, separation, detection, and semi-quantitation of physiologically-relevant peptide and protein hormones, such as AVP and ACTH, directly from human tissue. In addition, the relative simplicity, rapidity and specificity of the current methodology support the potential of this basic technology with further advancement for assisting surgical decision-making.« less

  9. Profiling of adrenocorticotropic hormone and arginine vasopressin in human pituitary gland and tumor thin tissue sections using droplet-based liquid-microjunction surface-sampling-HPLC–ESI-MS–MS

    SciTech Connect

    Kertesz, Vilmos; Calligaris, David; Feldman, Daniel R.; Changelian, Armen; Laws, Edward R.; Santagata, Sandro; Agar, Nathalie Y. R.; Van Berkel, Gary J.

    2015-06-18

    Described here are the results from the profiling of the proteins arginine vasopressin (AVP) and adrenocorticotropic hormone (ACTH) from normal human pituitary gland and pituitary adenoma tissue sections using a fully automated droplet-based liquid microjunction surface sampling-HPLC-ESI-MS/MS system for spatially resolved sampling, HPLC separation, and mass spectral detection. Excellent correlation was found between the protein distribution data obtained with this droplet-based liquid microjunction surface sampling-HPLC-ESI-MS/MS system and those data obtained with matrix assisted laser desorption ionization (MALDI) chemical imaging analyses of serial sections of the same tissue. The protein distributions correlated with the visible anatomic pattern of the pituitary gland. AVP was most abundant in the posterior pituitary gland region (neurohypophysis) and ATCH was dominant in the anterior pituitary gland region (adenohypophysis). The relative amounts of AVP and ACTH sampled from a series of ACTH secreting and non-secreting pituitary adenomas correlated with histopathological evaluation. ACTH was readily detected at significantly higher levels in regions of ACTH secreting adenomas and in normal anterior adenohypophysis compared to non-secreting adenoma and neurohypophysis. AVP was mostly detected in normal neurohypophysis as anticipated. This work demonstrates that a fully automated droplet-based liquid microjunction surface sampling system coupled to HPLC-ESI-MS/MS can be readily used for spatially resolved sampling, separation, detection, and semi-quantitation of physiologically-relevant peptide and protein hormones, such as AVP and ACTH, directly from human tissue. In addition, the relative simplicity, rapidity and specificity of the current methodology support the potential of this basic technology with further advancement for assisting surgical decision-making.

  10. Additional evidence for morpho-dimensional tooth crown variation in a New Indonesian H. erectus sample from the Sangiran Dome (Central Java).

    PubMed

    Zanolli, Clément

    2013-01-01

    This contribution reports fifteen human fossil dental remains found during the last two decades in the Sangiran Dome area, in Central Java, Indonesia. Among this sample, only one of the specimens had already been briefly described, with the other fourteen remaining unreported. Seven of the fifteen isolated teeth were found in a secured stratigraphic context in the late Lower-early Middle Pleistocene Kabuh Formation. The remaining elements were surface finds which, based on coincidental sources of information, were inferred as coming from the Kabuh Formation. Mainly constituted of permanent molars, but also including one upper incisor and one upper premolar, this dental sample brings additional evidence for a marked degree of size variation and time-related structural reduction in Javanese H. erectus. This is notably expressed by a significant decrease of the mesiodistal diameter, frequently associated to the reduction or even loss of the lower molar distal cusp (hypoconulid) and to a more square occlusal outline. In addition to the hypoconulid reduction or loss, this new sample also exhibits a low frequency of the occlusal Y-groove pattern, with a dominance of the X and, to a lesser extent, of the+patterns. This combination is rare in the Lower and early Middle Pleistocene paleoanthropological record, including in the early Javanese dental assemblage from the Sangiran Dome. On the other hand, similar dental features are found in Chinese H. erectus and in H. heidelbergensis. As a whole, this new record confirms the complex nature of the intermittent exchanges that occurred between continental and insular Southeast Asia through the Pleistocene.

  11. Additional Evidence for Morpho-Dimensional Tooth Crown Variation in a New Indonesian H. erectus Sample from the Sangiran Dome (Central Java)

    PubMed Central

    Zanolli, Clément

    2013-01-01

    This contribution reports fifteen human fossil dental remains found during the last two decades in the Sangiran Dome area, in Central Java, Indonesia. Among this sample, only one of the specimens had already been briefly described, with the other fourteen remaining unreported. Seven of the fifteen isolated teeth were found in a secured stratigraphic context in the late Lower-early Middle Pleistocene Kabuh Formation. The remaining elements were surface finds which, based on coincidental sources of information, were inferred as coming from the Kabuh Formation. Mainly constituted of permanent molars, but also including one upper incisor and one upper premolar, this dental sample brings additional evidence for a marked degree of size variation and time-related structural reduction in Javanese H. erectus. This is notably expressed by a significant decrease of the mesiodistal diameter, frequently associated to the reduction or even loss of the lower molar distal cusp (hypoconulid) and to a more square occlusal outline. In addition to the hypoconulid reduction or loss, this new sample also exhibits a low frequency of the occlusal Y-groove pattern, with a dominance of the X and, to a lesser extent, of the+patterns. This combination is rare in the Lower and early Middle Pleistocene paleoanthropological record, including in the early Javanese dental assemblage from the Sangiran Dome. On the other hand, similar dental features are found in Chinese H. erectus and in H. heidelbergensis. As a whole, this new record confirms the complex nature of the intermittent exchanges that occurred between continental and insular Southeast Asia through the Pleistocene. PMID:23843996

  12. Effect of surfactant addition on ultrasonic leaching of trace elements from plant samples in inductively coupled plasma-atomic emission spectrometry

    NASA Astrophysics Data System (ADS)

    Borkowska-Burnecka, Jolanta; Jankowiak, Urszula; Zyrnicki, Wieslaw; Anna Wilk, Kazimiera

    2004-04-01

    The applicability of surfactants in sample preparation of plant materials followed by analysis by inductively coupled plasma atomic emission spectrometry has been examined. Reference materials (INCT-MPH-2-Mixed Polish Herbs, INCT-TL-1 black tea leaves and CTA-VTL-2 -Virginia tobacco leaves) and commercially available tea leaves were analyzed. Effects of addition surfactants (Triton X-100, didodecyldimethylammonium bromide and cetyltrimethylammonium bromide) on efficiency of ultrasonic leaching of elements from the plant samples and on plasma parameters were investigated. Low concentrations of the surfactants in solutions did not affect, in practice, analytical line intensities and the nebulization process. Quantitative recovery of some elements could be obtained by ultrasonic diluted acid leaching with the aid of surfactants. However, the element recovery depended on type of surfactant, as well as element and sample material. Plasma parameters, i.e. the excitation temperatures of Ar I, Fe II and Ca II as well as the electron number density and the Mg II/Mg I intensity ratio did not vary significantly due to the surfactants in solutions.

  13. Collecting and Studying Blood and Tissue Samples From Patients With Locally Recurrent or Metastatic Prostate or Bladder/Urothelial Cancer

    ClinicalTrials.gov

    2016-12-06

    Healthy Control; Localized Urothelial Carcinoma of the Renal Pelvis and Ureter; Metastatic Malignant Neoplasm in the Bone; Metastatic Malignant Neoplasm in the Soft Tissues; Metastatic Urothelial Carcinoma of the Renal Pelvis and Ureter; Recurrent Bladder Carcinoma; Recurrent Prostate Carcinoma; Recurrent Urothelial Carcinoma of the Renal Pelvis and Ureter; Stage IV Bladder Cancer; Stage IV Bladder Urothelial Carcinoma; Stage IV Prostate Cancer

  14. Investigation of false positives associated with loop-mediated isothermal amplification assays for detection of Toxoplasma gondii in archived tissue samples of captive felids.

    PubMed

    Suleman, Essa; Mtshali, Moses Sibusiso; Lane, Emily

    2016-09-01

    Toxoplasma gondii is a ubiquitous protozoan parasite that infects humans and many different animals, including felids. Many molecular and serologic tests have been developed for detection of T. gondii in a wide range of hosts. Loop-mediated isothermal amplification (LAMP) is a field-friendly technique that lacks the practical drawbacks of other molecular and serologic tests, and LAMP assays have been successfully developed for detection of T. gondii in fresh tissue samples. In the current study, both a previously published and a de-novo designed LAMP assay were compared to a quantitative real-time (q)PCR assay, for the detection of T. gondii in archived formalin-fixed, paraffin-embedded (FFPE) tissue samples from captive wildlife. The LAMP assays produced conflicting results, generating both false positives and false negatives. Furthermore, the LAMP assays were unable to positively identify samples with low levels of parasites as determined by qPCR and histopathology. Therefore, these LAMP assays may not be the most suitable assays for detection of T. gondii in archived FFPE and frozen tissue samples.

  15. Determination of heroin and its main metabolites in small sample volumes of whole blood and brain tissue by reversed-phase liquid chromatography-tandem mass spectrometry.

    PubMed

    Karinen, Ritva; Andersen, Jannike Mørch; Ripel, Ase; Hasvold, Inger; Hopen, Anita Braute; Mørland, Jørg; Christophersen, Asbjørg S

    2009-09-01

    A high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed for the quantitative analysis of heroin and its major metabolites 6-acetylmorphine, morphine, morphine-3-glucuronide and morphine-6-glucuronide in blood and brain tissue, using 0.1-mL samples. We evaluated this method for analysis of heroin and its metabolites in samples from heroin treated mice. Ice-cold acidic buffer containing sodium fluoride was immediately added to blood and brain homogenate samples. Sample preparation was achieved by protein precipitation on ice-bath, using a mixture of ice-cold acetonitrile and methanol. The supernatant was evaporated to dryness, reconstituted with mobile phase, and injected into the chromatographic system. Separation was performed on a Xterra C18 column with gradient elution. The MS analysis was performed in positive ion mode, and multiple reaction monitoring (MRM) was used for drug quantification. The limits of quantification for the different opiates varied from 0.0007 to 0.02 mg/L in blood and from 0.002 to 0.06 microg/g in brain tissue. Day-to-day relative standard deviation ranged from 3.1 to 14.5%, and within-day variation ranged from 2.1 to 11.4%. The recoveries were between 80 and 111%. The stability of heroin was tested, and the study showed that heroin is more stable in brain tissue than in blood.

  16. LINE-1 Hypomethylation in Blood and Tissue Samples as an Epigenetic Marker for Cancer Risk: A Systematic Review and Meta-Analysis

    PubMed Central

    Barchitta, Martina; Quattrocchi, Annalisa; Maugeri, Andrea; Vinciguerra, Manlio; Agodi, Antonella

    2014-01-01

    Objective A systematic review and a meta-analysis were carried out in order to summarize the current published studies and to evaluate LINE-1 hypomethylation in blood and other tissues as an epigenetic marker for cancer risk. Methods A systematic literature search in the Medline database, using PubMed, was conducted for epidemiological studies, published before March 2014. The random-effects model was used to estimate weighted mean differences (MDs) with 95% Confidence Intervals (CIs). Furthermore, subgroup analyses were conducted by sample type (tissue or blood samples), cancer types, and by assays used to measure global DNA methylation levels. The Cochrane software package Review Manager 5.2 was used. Results A total of 19 unique articles on 6107 samples (2554 from cancer patients and 3553 control samples) were included in the meta-analysis. LINE-1 methylation levels were significantly lower in cancer patients than in controls (MD: −6.40, 95% CI: −7.71, −5.09; p<0.001). The significant difference in methylation levels was confirmed in tissue samples (MD −7.55; 95% CI: −9.14, −65.95; p<0.001), but not in blood samples (MD: −0.26, 95% CI: −0.69, 0.17; p = 0.23). LINE-1 methylation levels were significantly lower in colorectal and gastric cancer patients than in controls (MD: −8.33; 95% CI: −10.56, −6.10; p<0.001 and MD: −5.75; 95% CI: −7.75, −3.74; p<0.001) whereas, no significant difference was observed for hepatocellular cancer. Conclusions The present meta-analysis adds new evidence to the growing literature on the role of LINE-1 hypomethylation in human cancer and demonstrates that LINE-1 methylation levels were significantly lower in cancer patients than in control samples, especially in certain cancer types. This result was confirmed in tissue samples, both fresh/frozen or FFPE specimens, but not in blood. Further studies are needed to better clarify the role of LINE-1 methylation in specific subgroups, considering both cancer

  17. Application of non-lethal stable isotope analysis to assess feeding patterns of juvenile pallid sturgeon Scaphirhynchus albus: a comparison of tissue types and sample preservation methods

    USGS Publications Warehouse

    Andvik, R.T.; VanDeHey, J.A.; Fincel, M.J.; French, William E.; Bertrand, K.N.; Chipps, Steven R.; Klumb, R.A.; Graeb, B.D.S.

    2010-01-01

    Traditional techniques for stable isotope analysis (SIA) generally require sacrificing animals to collect tissue samples; this can be problematic when studying diets of endangered species such as the pallid sturgeon Scaphirhynchus albus. Our objectives were to (i) determine if pectoral fin tissue (non-lethal) could be a substitute for muscle tissue (lethal) in SIA of juvenile pallid sturgeon, and (ii) evaluate the influence of preservation techniques on stable isotope values. In the laboratory, individual juvenile pallid sturgeon were held for up to 186 day and fed chironomids, fish, or a commercially available pellet diet. Significant, positive relationships (r² ≥ 0.8) were observed between fin and muscle tissues for both δ15N and δ13C; in all samples isotopes were enriched in fins compared to muscle tissue. Chironomid and fish based diets of juvenile pallid sturgeon were distinguishable for fast growing fish (0.3 mm day−1) using stable δ15N and δ13C isotopes. Frozen and preserved fin tissue δ15N isotopes were strongly related (r2 = 0.89) but δ13C isotopes were weakly related (r2 = 0.16). Therefore, freezing is recommended for preservation of fin clips to avoid the confounding effect of enrichment by ethanol. This study demonstrates the utility of a non-lethal technique to assess time integrated food habits of juvenile pallid sturgeon and should be applicable to other threatened or endangered species.

  18. Proteomics analysis of tissue samples from patients with squamous cell carcinoma of the penis and positive to human papillomavirus

    PubMed Central

    Koifman, Leandro; Ornellas, Paulo; Ornellas, Antonio Augusto; Pereira, Denise de Abreu; Zingali, Benedeta Russolina; Cavalcanti, Silvia Maria Baeta; Afonso, Larissa Alves; Sandim, Vanessa; Alves, Gilda

    2015-01-01

    ABSTRACT Purpose: The aim of this study was to identify possible protein biomarkers and/or candidates for therapeutic targets in tissues of patients with SCCP, infected by HPV, applying one dimensional electrophoresis (1DE), followed by direct mass spectrometry (MS) analysis. Materials and Methods: Tissues from 10 HPV positive patients with SCCP and from 10 patients with HPV negative non-tumorous penile foreskins were analyzed applying 1D electrophoresis, followed by analysis with direct mass spectrometry (MS). Results: Sixty-three different proteins were identified in the first group and 50 in the second group. Recognition was possible for 28 proteins exclusively detected in Group 1 and 21 proteins presented only in Group 2. Conclusion: Some proteins in the first group are directly involved in the development of other types of cancer, and therefore, suitable for analysis. Complement C3 protein is a strong candidate for evaluating SCCP patients. PMID:26401855

  19. Maintaining Breast Cancer Specimen Integrity and Individual or Simultaneous Extraction of Quality DNA, RNA, and Proteins from Allprotect-Stabilized and Nonstabilized Tissue Samples

    PubMed Central

    Carroll, Paul; Donatello, Simona; Connolly, Elizabeth; Griffin, Mairead; Dunne, Barbara; Burke, Louise; Flavin, Richard; Rizkalla, Hala; Ryan, Ciara; Hayes, Brian; D'Adhemar, Charles; Banville, Niamh; Faheem, Nazia; Muldoon, Cian; Gaffney, Eoin F.

    2011-01-01

    The Saint James's Hospital Biobank was established in 2008, to develop a high-quality breast tissue BioResource, as a part of the breast cancer clinical care pathway. The aims of this work were: (1) to ascertain the quality of RNA, DNA, and protein in biobanked carcinomas and normal breast tissues, (2) to assess the efficacy of AllPrep® (Qiagen) in isolating RNA, DNA, and protein simultaneously, (3) to compare AllPrep with RNEasy® and QIAamp® (both Qiagen), and (4) to examine the effectiveness of Allprotect® (Qiagen), a new tissue stabilization medium in preserving DNA, RNA, and proteins. One hundred eleven frozen samples of carcinoma and normal breast tissue were analyzed. Tumor and normal tissue morphology were confirmed by frozen sections. Tissue type, tissue treatment (Allprotect vs. no Allprotect), extraction kit, and nucleic acid quantification were analyzed by utilizing a 4 factorial design (SPSS PASW 18 Statistics Software®). QIAamp (DNA isolation), AllPrep (DNA, RNA, and Protein isolation), and RNeasy (RNA isolation) kits were assessed and compared. Mean DNA yield and A260/280 values using QIAamp were 33.2 ng/μL and 1.86, respectively, and using AllPrep were 23.2 ng/μL and 1.94. Mean RNA yield and RNA Integrity Number (RIN) values with RNeasy were 73.4 ng/μL and 8.16, respectively, and with AllPrep were 74.8 ng/μL and 7.92. Allprotect-treated tissues produced higher RIN values of borderline significance (P=0.055). No discernible loss of RNA stability was detected after 6 h incubation of stabilized or nonstabilized tissues at room temperature or 4°C or in 9 freeze-thaw cycles. Allprotect requires further detailed evaluation, but we consider AllPrep to be an excellent option for the simultaneous extraction of RNA, DNA, and protein from tumor and normal breast tissues. The essential presampling procedures that maintain the diagnostic integrity of pathology specimens do not appear to compromise the quality of molecular isolates. PMID

  20. Maintaining Breast Cancer Specimen Integrity and Individual or Simultaneous Extraction of Quality DNA, RNA, and Proteins from Allprotect-Stabilized and Nonstabilized Tissue Samples.

    PubMed

    Mee, Blanaid C; Carroll, Paul; Donatello, Simona; Connolly, Elizabeth; Griffin, Mairead; Dunne, Barbara; Burke, Louise; Flavin, Richard; Rizkalla, Hala; Ryan, Ciara; Hayes, Brian; D'Adhemar, Charles; Banville, Niamh; Faheem, Nazia; Muldoon, Cian; Gaffney, Eoin F

    2011-12-01

    The Saint James's Hospital Biobank was established in 2008, to develop a high-quality breast tissue BioResource, as a part of the breast cancer clinical care pathway. The aims of this work were: (1) to ascertain the quality of RNA, DNA, and protein in biobanked carcinomas and normal breast tissues, (2) to assess the efficacy of AllPrep(®) (Qiagen) in isolating RNA, DNA, and protein simultaneously, (3) to compare AllPrep with RNEasy(®) and QIAamp(®) (both Qiagen), and (4) to examine the effectiveness of Allprotect(®) (Qiagen), a new tissue stabilization medium in preserving DNA, RNA, and proteins. One hundred eleven frozen samples of carcinoma and normal breast tissue were analyzed. Tumor and normal tissue morphology were confirmed by frozen sections. Tissue type, tissue treatment (Allprotect vs. no Allprotect), extraction kit, and nucleic acid quantification were analyzed by utilizing a 4 factorial design (SPSS PASW 18 Statistics Software(®)). QIAamp (DNA isolation), AllPrep (DNA, RNA, and Protein isolation), and RNeasy (RNA isolation) kits were assessed and compared. Mean DNA yield and A(260/280) values using QIAamp were 33.2 ng/μL and 1.86, respectively, and using AllPrep were 23.2 ng/μL and 1.94. Mean RNA yield and RNA Integrity Number (RIN) values with RNeasy were 73.4 ng/μL and 8.16, respectively, and with AllPrep were 74.8 ng/μL and 7.92. Allprotect-treated tissues produced higher RIN values of borderline significance (P=0.055). No discernible loss of RNA stability was detected after 6 h incubation of stabilized or nonstabilized tissues at room temperature or 4°C or in 9 freeze-thaw cycles. Allprotect requires further detailed evaluation, but we consider AllPrep to be an excellent option for the simultaneous extraction of RNA, DNA, and protein from tumor and normal breast tissues. The essential presampling procedures that maintain the diagnostic integrity of pathology specimens do not appear to compromise the quality of molecular isolates.

  1. Development of an indirect immunofluorescence assay for diagnosis of bovine viral diarrhoea virus on ear notch tissue samples in cattle infected persistently.

    PubMed

    Bedeković, Tomislav; Lemo, Nina; Lojkić, Ivana; Cvetnić, Zeljko; Cač, Zeljko; Madić, Josip

    2011-12-01

    Bovine viral diarrhoea virus (BVDV) causes a disease that has a wide range of clinical symptoms in domestic and wild ruminants. It is a major problem in cattle and causes significant economic losses in the cattle industry. The virus infects bovines of all ages and causes both immunosuppression and reproductive, respiratory and digestive disorders. Cattle infected persistently, as a continuing source of the virus and the main factor in transmission of the disease between and among herds, are the main source of BVDV and a primary factor in the epidemiology of the disease. To determine whether a BVDV infection is persistent, two samples should be taken at 3-4 week intervals and tested for the virus antigen. Animal sera, whole blood, organ and ear notch tissue samples can be used for BVDV diagnosis. In ear notch tissue, viral antigen can be detected by an antigen enzyme-linked immunosorbent assay (antigen ELISA), immunohistochemistry (IHC) and reverse-transcription polymerase chain reaction (RT-PCR). This paper describes the development and implementation of an indirect immunofluorescence (IF) method using ear notch tissue samples for diagnosis of cattle infected persistently. Results obtained by this method show that IF is a good alternative to RT-PCR and antigen ELISA and can be a quick and accurate method in diagnosis of BVDV in cattle infected persistently with this virus.

  2. Features and machine learning classification of connected speech samples from patients with autopsy proven Alzheimer's disease with and without additional vascular pathology.

    PubMed

    Rentoumi, Vassiliki; Raoufian, Ladan; Ahmed, Samrah; de Jager, Celeste A; Garrard, Peter

    2014-01-01

    Mixed vascular and Alzheimer-type dementia and pure Alzheimer's disease are both associated with changes in spoken language. These changes have, however, seldom been subjected to systematic comparison. In the present study, we analyzed language samples obtained during the course of a longitudinal clinical study from patients in whom one or other pathology was verified at post mortem. The aims of the study were twofold: first, to confirm the presence of differences in language produced by members of the two groups using quantitative methods of evaluation; and secondly to ascertain the most informative sources of variation between the groups. We adopted a computational approach to evaluate digitized transcripts of connected speech along a range of language-related dimensions. We then used machine learning text classification to assign the samples to one of the two pathological groups on the basis of these features. The classifiers' accuracies were tested using simple lexical features, syntactic features, and more complex statistical and information theory characteristics. Maximum accuracy was achieved when word occurrences and frequencies alone were used. Features based on syntactic and lexical complexity yielded lower discrimination scores, but all combinations of features showed significantly better performance than a baseline condition in which every transcript was assigned randomly to one of the two classes. The classification results illustrate the word content specific differences in the spoken language of the two groups. In addition, those with mixed pathology were found to exhibit a marked reduction in lexical variation and complexity compared to their pure AD counterparts.

  3. Differential Gene Expression of BRCA1,ERBB2 and TP53 biomarkers between Human Breast Tissue and Peripheral Blood Samples of Breast Cancer.

    PubMed

    Zghair, Abdulrazzaq Neamah; Sinha, Deepak Kumar; Kassim, Arkan; Alfaham, Mohmmad; Sharma, Anil K

    2016-01-01

    Breast cancer is a most common malignancy especially in Iraqi women accounting for high morbidity and mortality. Mutations in BRCA1 gene is one of the important genetic predisposing factors inbreast cancer. Similarly ERBB2 and TP53 are also key prognostic markers in breast cancer treatment.We were interested to explore the gene expression profiles of BRCA1, ERBB2 and TP53 in breast cancer women patients from Iraq so as to assess the potential of such markers in breast cancer treatment. The mRNA levels were significantly over-expressed in tumor tissues in comparison to normal ones with p values (p<0.005) observed between malignant BRCA1 and control tissue samples. Similarly significant difference (p<0.001) was observed between malignant ERBB2 in comparison to control, and malignant TP53 and benign tissue samples as well. However in blood samples, no considerable expression of these markers was observed. Out of three selected genes, ERBB2 expression was significantly expressed in comparison to BRCA1 and TP53 in cancer tissue. Mutation analysis of BRCA1, ERBB2 and TP53 has been made to find out the region most susceptible to mutations in these genes The BRCA1 exon 11, ERBB2 16 and TP53 exon 5 displayed increased chances of having mutations. We can conclude from the study that differential gene expression of BRCA1, ERBB2 and TP53 at mRNA levels may act as a diagnostic marker of circulating tumor cells having important prognostic value in breast cancer patients.

  4. Determination of selenium in marine biological tissues by transverse heated electrothermal atomic absorption spectrometry with longitudinal Zeeman background correction and automated ultrasonic slurry sampling.

    PubMed

    Méndez, H; Alava, F; Lavilla, I; Bendicho, C

    2001-01-01

    A fast, sensitive, and reliable method for determination of selenium in marine biological tissues by electrothermal atomic absorption spectrometry with slurry sampling was developed. Slurries were prepared from fresh and frozen seafood samples that were previously homogenized, dried, and ground; particle sizes <100 microm were taken for analysis. A 3% (v/v) HNO3 solution containing 0.01% (v/v) Triton X-100 was used as slurry diluent. Slurries were mixed on an automated ultrasonic slurry sampler at 20% amplitude for 30 s just before an aliquot was injected into the furnace. The method was successfully validated against the following certified reference materials: NRCC CRM DORM-2 (Dogfish muscle); NRCC CRM TORT-2 (Lobster hepatopancreas); NRCC CRM DOLT-2 (Dogfish liver); and BCR CRM 278 (Mussel tissue), and was subsequently applied to determination of Se in 10 marine biological samples. The influences of the drying procedure (oven-, microwave-, and freeze-drying), matrix modifier amount, mass of solid material in cup, and pipetting sequence are discussed. The limit of determination of Se was 0.16 microg/g and the repeatability, estimated as between-batch precision, was in the range of 4-8%. Se contents in the samples ranged from 0.6 to 2.8 microg/g. The proposed method should be useful for fast assessment of the daily dietary intake of Se.

  5. Genetic Characterization of Echinococcus granulosus from a Large Number of Formalin-Fixed, Paraffin-Embedded Tissue Samples of Human Isolates in Iran

    PubMed Central

    Rostami, Sima; Torbaghan, Shams Shariat; Dabiri, Shahriar; Babaei, Zahra; Mohammadi, Mohammad Ali; Sharbatkhori, Mitra; Harandi, Majid Fasihi

    2015-01-01

    Cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus, presents an important medical and veterinary problem globally, including that in Iran. Different genotypes of E. granulosus have been reported from human isolates worldwide. This study identifies the genotype of the parasite responsible for human hydatidosis in three provinces of Iran using formalin-fixed paraffin-embedded tissue samples. In this study, 200 formalin-fixed paraffin-embedded tissue samples from human CE cases were collected from Alborz, Tehran, and Kerman provinces. Polymerase chain reaction amplification and sequencing of the partial mitochondrial cytochrome c oxidase subunit 1 gene were performed for genetic characterization of the samples. Phylogenetic analysis of the isolates from this study and reference sequences of different genotypes was done using a maximum likelihood method. In total, 54.4%, 0.8%, 1%, and 40.8% of the samples were identified as the G1, G2, G3, and G6 genotypes, respectively. The findings of the current study confirm the G1 genotype (sheep strain) to be the most prevalent genotype involved in human CE cases in Iran and indicates the high prevalence of the G6 genotype with a high infectivity for humans. Furthermore, this study illustrates the first documented human CE case in Iran infected with the G2 genotype. PMID:25535316

  6. Real-time PCR strategy for parasite quantification in blood and tissue samples of experimental Trypanosoma cruzi infection.

    PubMed

    Caldas, Sérgio; Caldas, Ivo Santana; Diniz, Lívia de Figueiredo; Lima, Wanderson Geraldo de; Oliveira, Riva de Paula; Cecílio, Alzira Batista; Ribeiro, Isabela; Talvani, André; Bahia, Maria Terezinha

    2012-09-01

    The lack of an accurate diagnosis has been a serious obstacle to the advancement of the anti-Trypanosoma cruzi chemotherapy and long-term infection can result in different health risks to human. PCRs are alternative methods, more sensitive than conventional parasitological techniques, which due to their low sensitivities are considered unsuitable for these purposes. The aim of this study was to investigate a sensitive diagnostic strategy to quantify blood and cardiac tissues parasites based on real-time PCR tools during acute and chronic phases of murine Chagas disease, as well as to monitor the evolution of infection in those mice under specific treatment. In parallel, fresh blood examination, immunological analysis and quantification of cardiac inflammation were also performed to confront and improve real-time PCR data. Similar profiles of parasitemia curves were observed in both quantification techniques during the acute phase of the infection. In contrast, parasites could be quantified only by real-time PCR at 60 and 120 days of infection. In cardiac tissue, real-time PCR detected T. cruzi DNA in 100% of infected mice, and using this tool a significant Pearson correlation between parasite load in peripheral blood and in cardiac tissue during acute and chronic phases was observed. Levels of serum CCL2, CCL5 and nitric oxide were coincident with parasite load but focal and diffuse mononuclear infiltrates was observed, even with significant (p<0.05) reduction of parasitism after 60 days of infection. Later, this methodology was used to monitor the evolution of infection in animals treated with itraconazole (Itz). Itz-treatment induced a reduction of parasite load in both blood and cardiac muscle at the treatment period, but after the end of chemotherapy an increase of parasitism was detected. Interestingly, inflammatory mediators levels and heart inflammation intensity had similar evolution to the parasite load, in the group of animals treated. Taken together, our

  7. A correlative method for imaging identical regions of samples by micro-CT, light microscopy, and electron microscopy: imaging adipose tissue in a model system.

    PubMed

    Sengle, Gerhard; Tufa, Sara F; Sakai, Lynn Y; Zulliger, Martin A; Keene, Douglas R

    2013-04-01

    We present a method in which a precise region of interest within an intact organism is spatially mapped in three dimensions by non-invasive micro-computed X-ray tomography (micro-CT), then further evaluated by light microscopy (LM) and transmission electron microscopy (TEM). Tissues are prepared as if for TEM including osmium fixation, which imparts soft tissue contrast in the micro-CT due to its strong X-ray attenuation. This method may therefore be applied to embedded, archived TEM samples. Upon selection of a two-dimensional (2-D) projection from a region of interest (ROI) within the three-dimensional volume, the epoxy-embedded sample is oriented for microtomy so that the sectioning plane is aligned with the micro-CT projection. Registration is verified by overlaying LM images with 2-D micro-CT projections. Structures that are poorly resolved in the micro-CT may be evaluated at TEM resolution by observing the next serial ultrathin section, thereby accessing the same ROI by all three imaging techniques. We compare white adipose tissue within the forelimbs of mice harboring a lipid-altering mutation with their littermate controls. We demonstrate that individual osmium-stained lipid droplets as small as 15 µm and separated by as little as 35 µm may be discerned as separate entities in the micro-CT, validating this to be a high-resolution, non-destructive technique for evaluation of fat content.

  8. Results of quality-control sampling of water, bed sediment, and tissue in the Western Lake Michigan Drainages study unit of the National Water-Quality Assessment Program

    USGS Publications Warehouse

    Fitzgerald, S.A.

    1997-01-01

    This report contains the quality control results of the Western Lake Michigan Drainages study unit of the National Water Quality Assessment Program. Quality control samples were collected in the same manner and contemporaneously with environmental samples during the first highintensity study phase in the unit (1992 through 1995) and amounted to approximately 15 percent of all samples collected. The accuracy and precision of hundreds of chemical analyses of surface and ground-water, bed sediment, and tissue was determined through the collection and analysis of field blanks, field replicates and splits, matrix spikes, and surrogates. Despite the several detections of analytes in the field blanks, the concentrations of most constituents in the environmental samples will likely be an order of magnitude or higher than those in the blanks. However, frequent detections, and high concentrations, of dissolved organic carbon (DOC) in several surface and ground-water blanks are probably significant with respect to commonly measured environmental concentrations, and the environmental data will have to be qualified accordingly. The precision of sampling of water on a percent basis, as determined from replicates and splits, was generally proportional to the concentration of the constituents, with constituents present in relatively high concentrations generally having less sampling variability than those with relatively low concentrations. In general, analytes with relatively high variability between replicates were present at concentrations near the reporting limit or were associated with relatively small absolute concentration differences, or both. Precision of replicates compared to that for splits in bed sediment samples was similar, thus eliminating sampling as a major source of variability in analyte concentrations. In the case the phthalates in bed sediment, contamination in either the field or laboratory could have caused the relatively large variability between replicate

  9. Comparison of tissue and fluid samples for the early detection of canine distemper virus in experimentally infected dogs.

    PubMed

    Kim, Doo; Jeoung, Seok-Yong; Ahn, So-Jeo; Lee, Jong-Hyun; Pak, Son-Il; Kwon, Hyuk-Moo

    2006-08-01

    The clinical utility of various specimens was examined for the early diagnosis of canine distemper (CD). Seven healthy dogs at 17 weeks of age were experimentally infected with a field isolate of canine distemper virus. The RT-PCR was carried out to detect CDV NP gene. Dogs showed mild fever and leukopenia, however, typical clinical signs of CD were not seen through the experimental period. CDV amplicons were detected more, earlier and for longer period in the conjunctival swabs than in the other samples employed. These results suggested that conjunctival swab samples, which are easy to obtain and non-invasive, would be the most suitable and practical specimen for the early antemortem diagnosis of CDV infection.

  10. A rapid analytical method to quantify complex organohalogen contaminant mixtures in large samples of high lipid mammalian tissues.

    PubMed

    Desforges, Jean-Pierre; Eulaers, Igor; Periard, Luke; Sonne, Christian; Dietz, Rune; Letcher, Robert J

    2017-06-01

    In vitro investigations of the health impact of individual chemical compounds have traditionally been used in risk assessments. However, humans and wildlife are exposed to a plethora of potentially harmful chemicals, including organohalogen contaminants (OHCs). An alternative exposure approach to individual or simple mixtures of synthetic OHCs is to isolate the complex mixture present in free-ranging wildlife, often non-destructively sampled from lipid rich adipose. High concentration stock volumes required for in vitro investigations do, however, pose a great analytical challenge to extract sufficient amounts of complex OHC cocktails. Here we describe a novel method to easily, rapidly and efficiently extract an environmentally accumulated and therefore relevant contaminant cocktail from large (10-50 g) marine mammal blubber samples. We demonstrate that lipid freeze-filtration with acetonitrile removes up to 97% of blubber lipids, with minimal effect on the efficiency of OHC recovery. Sample extracts after freeze-filtration were further processed to remove residual trace lipids via high-pressure gel permeation chromatography and solid phase extraction. Average recoveries of OHCs from triplicate analysis of killer whale (Orcinus orca), polar bear (Ursus maritimus) and pilot whale (Globicephala spp.) blubber standard reference material (NIST SRM-1945) ranged from 68 to 80%, 54-92% and 58-145%, respectively, for (13)C-enriched internal standards of six polychlorinated biphenyl congeners, 16 organochlorine pesticides and four brominated flame retardants. This approach to rapidly generate OHC mixtures shows great potential for experimental exposures using complex contaminant mixtures, research or monitoring driven contaminant quantification in biological samples, as well as the untargeted identification of emerging contaminants.

  11. Semiempirical Rules To Determine Drug Sensitivity and Ionization Efficiency in Secondary Ion Mass Spectrometry Using a Model Tissue Sample.

    PubMed

    Vorng, Jean-Luc; Kotowska, Anna M; Passarelli, Melissa K; West, Andrew; Marshall, Peter S; Havelund, Rasmus; Seah, Martin P; Dollery, Colin T; Rakowska, Paulina D; Gilmore, Ian S

    2016-11-15

    There is an increasing need in the pharmaceutical industry to reduce drug failure at late stage and thus reduce the cost of developing a new medicine. Since most drug targets are intracellular, this requires a better understanding of the drug disposition within a cell. Secondary ion mass spectrometry has been identified as a potentially important technique to do this, as it is label-free and allows imaging in 3D with subcellular resolution and recent studies have shown promise for amiodarone. An important analytical parameter is sensitivity, and we measure this in a bovine liver homogenate reference sample for 20 drugs representing important class types relevant to the pharmaceutical industry. We also measure the sensitivity for pure drug and show, for the first time, that the secondary ion mass spectrometry (SIMS) positive ionization efficiency for small molecules is a simple power-law relationship to the log P value. This discovery will be important for advancing the understanding of the SIMS ionization process in small molecules that has, until now, been elusive. This simple relationship is found to hold true for drug doped in the bovine liver homogenate reference sample, except for fluticasone, nicardipine, and sorafenib which suffer from severe matrix suppression. This relationship provides a simple semiempirical method to determine drug sensitivity for positive secondary ions. Furthermore, we show, on chosen models, how the use of different solvents during sample preparation can affect the ionization of analytes.

  12. Monte Carlo simulations incorporating Mie calculations of light transport in tissue phantoms: Examination of photon sampling volumes for endoscopically compatible fiber optic probes

    SciTech Connect

    Mourant, J.R.; Hielscher, A.H.; Bigio, I.J.

    1996-04-01

    Details of the interaction of photons with tissue phantoms are elucidated using Monte Carlo simulations. In particular, photon sampling volumes and photon pathlengths are determined for a variety of scattering and absorption parameters. The Monte Carlo simulations are specifically designed to model light delivery and collection geometries relevant to clinical applications of optical biopsy techniques. The Monte Carlo simulations assume that light is delivered and collected by two, nearly-adjacent optical fibers and take into account the numerical aperture of the fibers as well as reflectance and refraction at interfaces between different media. To determine the validity of the Monte Carlo simulations for modeling the interactions between the photons and the tissue phantom in these geometries, the simulations were compared to measurements of aqueous suspensions of polystyrene microspheres in the wavelength range 450-750 nm.

  13. Ultrasound-assisted extraction followed by disposable pipette purification for the determination of polychlorinated biphenyls in small-size biological tissue samples.

    PubMed

    Pena-Abaurrea, M; García de la Torre, V S; Ramos, L

    2013-11-22

    The use of solid-phase extraction pipette tip (also called disposable pipette extraction, DPX) has been evaluated for the purification of environmentally relevant polychlorinated biphenyls (PCBs) in fatty extracts obtained by ultrasound-assisted extraction with a sonication probe from small-size biological tissues. Complete sample treatment involved only 50 mg of sample and was completed in ca. 15 min with minimal sample manipulation and reagents consumption (i.e., 1.5 mL of n-hexane and 0.8 g of acidic silica). The performance of the proposed methodology for the intended determination was firstly evaluated by determination of the endogenous PCB levels in a naturally contaminated internal reference material. The determined concentrations showed a good agreement with those obtained using a more conventional sample preparation procedure previously validated in our laboratory (recoveries, as compared to levels determined using the latter method, were in the 85-123% range for a large majority of the studied congeners, and the relative standard deviations were in general lower than 14%). Results obtained for the analysis of reference food samples and certified reference materials NIST 1945 and 1947 demonstrated that, when combined with gas chromatography coupled to ion trap mass spectrometry working in the tandem mode, GC-ITD(MS/MS), the proposed methodology allowed accurate determination of most of the investigated PCBs and that 50 mg of sample sufficed for the screening of less abundant toxic congeners.

  14. Quantitative determination of cetirizine enantiomers in guinea pig plasma, brain tissue and microdialysis samples using liquid chromatography/tandem mass spectrometry.

    PubMed

    Gupta, Anubha; Jansson, Britt; Chatelain, Pierre; Massingham, Roy; Hammarlund-Udenaes, Margareta

    2005-01-01

    Sensitive enantioselective liquid chromatographic assays using tandem mass spectrometric detection were developed and validated for the determination of S-cetirizine (S-CZE) and R-cetirizine (R-CZE) in guinea pig plasma, brain tissue, and microdialysis samples. Enantioselective separation was achieved on an alpha1-acid glycoprotein column within 14 min for all methods. A cetirizine analog, ucb 20028, was used as internal standard. Cetirizine and the internal standard were detected by multiple reaction monitoring using transitions m/z 389.1 --> 200.9 and 396.1 --> 276.1, respectively. The samples were prepared using protein precipitation with acetonitrile. For guinea pig plasma, the assay was linear over the range 0.25-5000 ng/mL for both S-CZE and R-CZE, with a lower limit of quantification (LLOQ) of 0.25 ng/mL. For the brain tissue and microdialysis samples, the assays were linear over the range 2.5-250 ng/g and 0.25-50 ng/mL, respectively, and the LLOQ values were 2.5 ng/g and 0.25 ng/mL, respectively. The intra- and inter-day precision values were < or =7.1% and < or =12.6%, respectively, and the intra- and inter-day accuracy varied by less than +/-8.0% and +/-6.0% of the nominal value, respectively, for both enantiomers in all the matrices investigated.

  15. Tissue and serum samples of patients with papillary thyroid cancer with and without benign background demonstrate different altered expression of proteins

    PubMed Central

    Abdullah, Mardiaty Iryani; Lee, Ching Chin; Mat Junit, Sarni; Ng, Khoon Leong

    2016-01-01

    Background Papillary thyroid cancer (PTC) is mainly diagnosed using fine-needle aspiration biopsy. This most common form of well-differentiated thyroid cancer occurs with or without a background of benign thyroid goiter (BTG). Methods In the present study, a gel-based proteomics analysis was performed to analyse the expression of proteins in tissue and serum samples of PTC patients with (PTCb; n = 6) and without a history of BTG (PTCa; n = 8) relative to patients with BTG (n = 20). This was followed by confirmation of the levels of proteins which showed significant altered abundances of more than two-fold difference (p < 0.01) in the tissue and serum samples of the same subjects using ELISA. Results The data of our study showed that PTCa and PTCb distinguish themselves from BTG in the types of tissue and serum proteins of altered abundance. While higher levels of alpha-1 antitrypsin (A1AT) and heat shock 70 kDa protein were associated with PTCa, lower levels of A1AT, protein disulfide isomerase and ubiquitin-conjugating enzyme E2 N seemed apparent in the PTCb. In case of the serum proteins, higher abundances of A1AT and alpha 1-beta glycoprotein were detected in PTCa, while PTCb was associated with enhanced apolipoprotein A-IV and alpha 2-HS glycoprotein (AHSG). The different altered expression of tissue and serum A1AT as well as serum AHSG between PTCa and PTCb patients were also validated by ELISA. Discussion The distinctive altered abundances of the tissue and serum proteins form preliminary indications that PTCa and PTCb are two distinct cancers of the thyroid that are etiologically and mechanistically different although it is currently not possible to rule out that they may also be due other reasons such as the different stages of the malignant disease. These proteins stand to have a potential use as tissue or serum biomarkers to discriminate the three different thyroid neoplasms although this requires further validation in clinically representative

  16. A lab-on-a-chip system integrating tissue sample preparation and multiplex RT-qPCR for gene expression analysis in point-of-care hepatotoxicity assessment.

    PubMed

    Lim, Geok Soon; Chang, Joseph S; Lei, Zhang; Wu, Ruige; Wang, Zhiping; Cui, Kemi; Wong, Stephen

    2015-10-21

    A truly practical lab-on-a-chip (LOC) system for point-of-care testing (POCT) hepatotoxicity assessment necessitates the embodiment of full-automation, ease-of-use and "sample-in-answer-out" diagnostic capabilities. To date, the reported microfluidic devices for POCT hepatotoxicity assessment remain rudimentary as they largely embody only semi-quantitative or single sample/gene detection capabilities. In this paper, we describe, for the first time, an integrated LOC system that is somewhat close to a practical POCT hepatotoxicity assessment device - it embodies both tissue sample preparation and multiplex real-time RT-PCR. It features semi-automation, is relatively easy to use, and has "sample-in-answer-out" capabilities for multiplex gene expression analysis. Our tissue sample preparation module incorporating both a microhomogenizer and surface-treated paramagnetic microbeads yielded high purity mRNA extracts, considerably better than manual means of extraction. A primer preloading surface treatment procedure and the single-loading inlet on our multiplex real-time RT-PCR module simplify off-chip handling procedures for ease-of-use. To demonstrate the efficacy of our LOC system for POCT hepatotoxicity assessment, we perform a preclinical animal study with the administration of cyclophosphamide, followed by gene expression analysis of two critical protein biomarkers for liver function tests, aspartate transaminase (AST) and alanine transaminase (ALT). Our experimental results depict normalized fold changes of 1.62 and 1.31 for AST and ALT, respectively, illustrating up-regulations in their expression levels and hence validating their selection as critical genes of interest. In short, we illustrate the feasibility of multiplex gene expression analysis in an integrated LOC system as a viable POCT means for hepatotoxicity assessment.

  17. Body fluid and tissue analysis using filter paper sampling support prior to LC-MS/MS: application to fatal overdose with colchicine.

    PubMed

    Lauer, Estelle; Widmer, Christèle; Versace, François; Staub, Christian; Mangin, Patrice; Sabatasso, Sara; Augsburger, Marc; Déglon, Julien

    2013-01-01

    Because of the various matrices available for forensic investigations, the development of versatile analytical approaches allowing the simultaneous determination of drugs is challenging. The aim of this work was to assess a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform allowing the rapid quantification of colchicine in body fluids and tissues collected in the context of a fatal overdose. For this purpose, filter paper was used as a sampling support and was associated with an automated 96-well plate extraction performed by the LC autosampler itself. The developed method features a 7-min total run time including automated filter paper extraction (2 min) and chromatographic separation (5 min). The sample preparation was reduced to a minimum regardless of the matrix analyzed. This platform was fully validated for dried blood spots (DBS) in the toxic concentration range of colchicine. The DBS calibration curve was applied successfully to quantification in all other matrices (body fluids and tissues) except for bile, where an excessive matrix effect was found. The distribution of colchicine for a fatal overdose case was reported as follows: peripheral blood, 29 ng/ml; urine, 94 ng/ml; vitreous humour and cerebrospinal fluid, < 5 ng/ml; pericardial fluid, 14 ng/ml; brain, < 5 pg/mg; heart, 121 pg/mg; kidney, 245 pg/mg; and liver, 143 pg/mg. Although filter paper is usually employed for DBS, we report here the extension of this alternative sampling support to the analysis of other body fluids and tissues. The developed platform represents a rapid and versatile approach for drug determination in multiple forensic media.

  18. Development of a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of phenylethanolamine A in tissue and feed samples and confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS).

    PubMed

    Cao, Biyun; He, Guangzhao; Yang, Hong; Chang, Huafang; Li, Shuqun; Deng, Anping

    2013-10-15

    Phenylethanolamine A (PA) is a new emerged β-adrenergic agonist illegally used as feed additives for growth promotion. In this study, a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of PA in tissue and feed samples was developed and confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS). By reduction of nitryl group to amino group, the PA derivative was synthesized and coupled to carrier proteins with diazobenzidine method. The antisera obtained from four immunized rabbits were characterized in terms of sensitivity and specificity. All antisera displayed high sensitivity with IC50 values lower than 0.48 ng mL(-1). The most sensitive ELISA was established with IC50 and limit of detection (LOD) values of 0.049 ng mL(-1) and 0.003 ng mL(-1), respectively. The cross-reactivity (CR) values of the antisera with three frequently used β-adrenergic agonists (clenbuterol, salbutamol and ractopamine) were lesser than 0.39%; there was no CR of the antisera with other six compounds including two structurally related substances (isoproterenol, phenylephrine). To investigate the accuracy and precision of the assay, swine kidney, liver, meat and feed samples were fortified with PA at different content and analyzed by ELISA. Acceptable recovery rates of 92.2-113.7% and intra-assay coefficients of variation of 3.8-10.9% (n=3) were achieved. Seven spiked samples were simultaneously analyzed by ELISA and LC-MS/MS. There was a high correlation coefficient of 0.9956 (n=7) between the two methods. The proposed ELISA proven to be a feasible quantitative/screening method for PA analysis in tissue and feed samples with the properties of high sensitivity and specificity, high sample throughput and low expensive.

  19. Variations of midfacial soft-tissue thickness in subjects aged between 6 and 18years for the reconstruction of the profile: A study on an Italian sample.

    PubMed

    Gibelli, Daniele; Collini, Federica; Porta, Davide; Zago, Matteo; Dolci, Claudia; Cattaneo, Cristina; Sforza, Chiarella

    2016-09-01

    Facial approximation techniques used in forensic anthropology are based on soft-tissue thickness databases. However most of articles deal with adult populations, and very few studies were conducted on minors. This study aims at providing data concerning facial thickness for George reconstruction, preliminary to the 3D facial approximation. Diagnostic cephalometric X-ray films were obtained from 222 healthy Caucasoid children (91 boys and 131 girls), aged between 6 and 18years, treated in a Department of Orthodontics of Northern Italy. After setting the Frankfurt plane horizontal, 14 measurements were taken at the mid-facial landmarks: supraglabella, glabella, nasion, nasale, subnasale, superior labial sulcus, labrale superius, stomion, labrale inferius, inferior labial sulcus, suprapogonion, pogonion, gnathion, menton. Mean and standard deviation of soft-tissues thickness at each point were calculated. A two-way analysis of variance (ANOVA) was performed to test the modifications of facial parameters with age and sex (p<0.01). The results demonstrated that there is an increase in tissue thickness as individuals grow; in most occasions, males showed thicker soft tissues than females of the same age, especially after the adolescent growth spurt. Facial thicknesses at subnasale, superior labial sulcus, labrale superius, labrale inferius, inferior labial sulcus, suprapogonion, pogonion and gnathion significantly modified with age, whereas the same parameters at subnasale, superior labial sulcus, labrale superius, labrale inferius, stomion and suprapogonion were significantly sexually dimorphic. In addition, a database for soft-tissue thicknesses in children aged between 6 and 18years was created, which may be of interest in cases of facial approximation of Italian minors.

  20. Sensitive Mie scattering immunoagglutination assay of porcine reproductive and respiratory syndrome virus (PRRSV) from lung tissue samples in a microfluidic chip.

    PubMed

    Song, Jae-Young; Lee, Chang-Hee; Choi, Eun-Jin; Kim, Keesung; Yoon, Jeong-Yeol

    2011-12-01

    A microfluidic immunosensor utilizing Mie scattering immunoaggultination assay was developed for rapid and sensitive detection of porcine reproductive and respiratory syndrome virus (PRRSV) from lung tissue samples of domesticated pigs. Antibodies against PRRSV were conjugated to the surface of highly carboxylated polystyrene microparticles (diameter=920nm) and mixed with the diluted PRRSV tissue samples in a Y-shaped microchannel. Antibody-antigen binding induced microparticle immunoagglutination, which was detected by measuring the forward 45° light scattering of 380nm incident beam using microcallipered, proximity fiber optics. For comparison, multi-well experiments were also performed using the same optical detection setup. The detection limit was determined to be 10(-3)TCID(50)ml(-1) for PRRSV dissolved in PBS, while those of previous RT-PCR studies for PRRSV were 10(1)TCID(50)ml(-1) (conventional assays) or <1TCID(50)ml(-1) (quantitative real-time assays). Mie scattering simulations were able to predict the shape of the PRRSV standard curve, indicating that any non-linearity of the standard curve can be interpreted purely as an optical phenomenon. Each assay took less than 5min. A strong correlation could be found between RT-PCR and this method for the lung tissue samples, even though their respective detection mechanisms are different fundamentally (nucleic acids for RT-PCR and virus antigens for light scattering immunoagglutination assay). Several different dilution factors were also tested for tissue samples, and 1/10 and 1/100 were found to be usable. If the microfluidic chips are used only once (i.e. without re-using them), both superior sensitivity and satisfactory specificity can be demonstrated. Specificity studies revealed the presence of Type II PRRSV and non-presence of Type I PRRSV and that the microfluidic chip assay could detect Type II North American strain of PRRSV for the animals tested. This work demonstrates the potential of the Mie

  1. Identification of Specific miRNA Signature in Paired Sera and Tissue Samples of Indian Women with Triple Negative Breast Cancer

    PubMed Central

    Thakur, Seema; Grover, Rajesh K.; Gupta, Sanjay; Yadav, Ajay K.; Das, Bhudev C.

    2016-01-01

    Of several subtypes of breast cancer, triple negative breast cancer (TNBC) is a highly aggressive tumor that lacks expression of hormone receptors for estrogen, progesterone and human epidermal growth factor receptor 2 and shows a worst prognosis. The small noncoding RNAs (miRNAs) considered as master regulator of gene expression play a key role in cancer initiation, progression and drug resistance and have emerged as attractive molecular biomarkers for diagnosis, prognosis and treatment targets in cancer. We have done expression profiling of selected miRNAs in paired serum and tissue samples of TNBC patients and corresponding cell lines and compared with that of other subtypes, in order to identify novel serum miRNA biomarkers for early detection and progression of TNBC. A total of 85 paired tumor tissues and sera with an equal number of adjacent normal tissue margins and normal sera from age matched healthy women including tissue and sera samples from 15 benign fibroadenomas were employed for the study. We report for the first time an extremely high prevalence (73.9%) of TNBC in premenopausal women below 35 years of age and a significant altered expression of a panel of three specific oncogenic miRNAs- miR-21, miR-221, miR-210, and three tumor suppressor miRNAs- miR-195, miR-145 and Let-7a in both tissues and corresponding sera of TNBC patients when compared with triple positive breast cancer (TPBC) patients. While miR-21, miR-221 and miR-210 showed significant over-expression, miR-195 and miR-145 were downregulated and well correlated with various clinicopathological and demographic risk factors, tumor grade, clinical stage and hormone receptor status. Interestingly, despite being a known tumor suppressor, Let-7a showed a significant overexpression in TNBCs. It is suggested that this panel of six miRNA signature may serve as a minimally invasive biomarker for an early detection of TNBC patients. PMID:27404381

  2. Simplified matrix solid phase dispersion procedure for the determination of parabens and benzophenone-ultraviolet filters in human placental tissue samples.

    PubMed

    Vela-Soria, F; Rodríguez, I; Ballesteros, O; Zafra-Gómez, A; Ballesteros, L; Cela, R; Navalón, A

    2014-12-05

    In recent decades, the industrial development has resulted in the appearance of a large amount of new chemicals that are able to produce disorders in the human endocrine system. These substances, so-called endocrine disrupting chemicals (EDCs), include many families of compounds, such as parabens and benzophenone-UV filters. Taking into account the demonstrated biological activity of these compounds, it is necessary to develop new analytical procedures to assess the exposure in order to establish, in an accurate way, relationships between EDCs and harmful health effects in population. In the present work, a new method based on a simplified sample treatment by matrix solid phase dispersion (MSPD) followed by ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis, is validated for the determination of four parabens (methyl-, ethyl-, propyl- and butylparaben) and six benzophenone-UV filters (benzophenone-1, benzophenone-2, benzophenone-3, benzophenone-6, benzophenone-8 and 4-hydroxybenzophenone) in human placental tissue samples. The extraction parameters were accurately optimized using multivariate optimization strategies. Ethylparaben ring-13C6 and benzophenone-d10 were used as surrogates. The found limits of quantification ranged from 0.2 to 0.4 ng g(-1) and inter-day variability (evaluated as relative standard deviation) ranged from 5.4% to 12.8%. The method was validated using matrix-matched standard calibration followed by a recovery assay with spiked samples. Recovery rates ranged from 96% to 104%. The method was satisfactorily applied for the determination of compounds in human placental tissue samples collected at the moment of delivery from 10 randomly selected women.

  3. Analysis of organochlorines in harbor seal (Phoca vitulina) tissue samples from Alaska using gas chromatography/ion trap mass spectrometry by an isotopic dilution technique.

    PubMed

    Wang, Dongli; Atkinson, Shannon; Hoover-Miller, Anne; Li, Qing X

    2005-01-01

    A gas chromatography/ion trap mass spectrometry (GC/ITMS) method was developed for the determination of organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs) and polychlorinated naphthalenes (PCNs) in harbor seal (Phoca vitulina) tissues. Tissue samples were homogenized, lyophilized and fortified with (13)C-PCBs 28, 123, 169 and 170, and then extracted with an accelerated solvent extractor with a mixture of hexane and methylene chloride (1:1, v/v). After lipid removal using a 40% H(2)SO(4)-modified silica gel column, all organochlorines were collected in one fraction and further fractionated with an activated carbon/silica gel (1:20) column into a first fraction containing OCPs, non-coplanar PCBs and (13)C-PCBs 28, 123 and 170, and a second containing PCNs, coplanar PCBs and (13)C-PCB 169. Prior to GC/MS/MS analysis, (13)C-PCB 169 was added into the first fraction as an injection standard and (13)C-PCB 170 into the second fraction to calibrate the recoveries of the fortified internal standards. This method can effectively eliminate matrix interferences, and has high selectivity and sensitivity. Recoveries averaged 45-86% for OCPs with relative standard deviations (RSDs) of 2-14%, 52-137% for PCBs with RSDs of 3-29% and 36-152% for PCNs with RSDs of 7-29% from lard and chicken heart samples, which were used as alternative matrices to harbor seal samples in recovery studies. The limits of detection for OCPs, PCBs and PCNs were 0.7-1.9, 1.5-8.9 and 0.5-10 pg/g dry weight, respectively. This method can be used to analyze OCPs, PCBs and PCNs in harbor seal blubber, liver and kidney samples.

  4. Precise simultaneous quantification of methadone and cocaine in rat serum and brain tissue samples following their successive i.p. administration.

    PubMed

    Nakhla, David S; Hussein, Lobna A; Magdy, N; Abdallah, Inas A; Hassan, Hazem E

    2017-03-24

    A sensitive high-performance liquid chromatography (HPLC) assay with dual UV detection has been developed and validated for the simultaneous quantification of methadone and cocaine in rat serum and brain tissue samples. Liquid-liquid extraction using hexanes was applied for samples extraction with Levo-Tetrahydropalmatine (L-THP) as the internal standard. Chromatographic separation of the analytes was achieved on a reversed-phase Waters Symmetry(®) C18 column (150mm×4.6mm, 5μm). A gradient elution was employed with a mobile phase consisting of 5mM potassium phosphate containing 0.1% triethylamine (pH=6.5) (A) and acetonitrile (B) with a flow rate of 1mL/min. UV detection was employed at 215nm and 235nm for the determination of methadone and cocaine, respectively. The calibration curves were linear over the range of 0.05-10μg/mL for both methadone and cocaine. The assay was validated according to FDA guidelines for bioanalytical method validation and results were satisfactory and met FDA criteria. Inter-day accuracy values of serum and brain samples ranged from 96.97 to 105.59% while intra-day accuracy values ranged from 91.49 to 111.92%. Stability assays showed that both methadone and cocaine were stable during sample storage, preparation, and analytical procedures. The method was successfully used to analyze biological samples obtained from a drug- drug interaction pharmacokinetics (PK) study conducted in rats to investigate the effect of methadone on cocaine PK. Our method not only can be used for bioanalysis of samples obtained from rats but also can potentially be applied to human biological serum samples to monitor compliance to methadone maintenance therapy (MMT) and to detect possible cocaine-methadone co-abuse.

  5. Virome profiling of bats from Myanmar by metagenomic analysis of tissue samples reveals more novel Mammalian viruses.

    PubMed

    He, Biao; Li, Zuosheng; Yang, Fanli; Zheng, Junfeng; Feng, Ye; Guo, Huancheng; Li, Yingying; Wang, Yiyin; Su, Nan; Zhang, Fuqiang; Fan, Quanshui; Tu, Changchun

    2013-01-01

    Bats are reservoir animals harboring many important pathogenic viruses and with the capability of transmitting these to humans and other animals. To establish an effective surveillance to monitor transboundary spread of bat viruses between Myanmar and China, complete organs from the thorax and abdomen from 853 bats of six species from two Myanmar counties close to Yunnan province, China, were collected and tested for their virome through metagenomics by Solexa sequencing and bioinformatic analysis. In total, 3,742,314 reads of 114 bases were generated, and over 86% were assembled into 1,649,512 contigs with an average length of 114 bp, of which 26,698 (2%) contigs were recognizable viral sequences belonging to 24 viral families. Of the viral contigs 45% (12,086/26,698) were related to vertebrate viruses, 28% (7,443/26,698) to insect viruses, 27% (7,074/26,698) to phages and 95 contigs to plant viruses. The metagenomic results were confirmed by PCR of selected viruses in all bat samples followed by phylogenetic analysis, which has led to the discovery of some novel bat viruses of the genera Mamastrovirus, Bocavirus, Circovirus, Iflavirus and Orthohepadnavirus and to their prevalence rates in two bat species. In conclusion, the present study aims to present the bat virome in Myanmar, and the results obtained further expand the spectrum of viruses harbored by bats.

  6. Prediction of Recurrence and Survival for Triple-Negative Breast Cancer (TNBC) by a Protein Signature in Tissue Samples.

    PubMed

    Campone, Mario; Valo, Isabelle; Jézéquel, Pascal; Moreau, Marie; Boissard, Alice; Campion, Loic; Loussouarn, Delphine; Verriele, Véronique; Coqueret, Olivier; Guette, Catherine

    2015-11-01

    To date, there is no available targeted therapy for patients who are diagnosed with triple-negative breast cancers (TNBC). The aim of this study was to identify a new specific target for specific treatments. Frozen primary tumors were collected from 83 adjuvant therapy-naive TNBC patients. These samples were used for global proteome profiling by iTRAQ-OFFGEL-LC-MS/MS approach in two series: a training cohort (n = 42) and a test set (n = 41). Patients who remains free of local or distant metastasis for a minimum of 5 years after surgery were classified in the no-relapse group; the others were in the relapse group. OPLS and Kaplan-Meier analyses were performed to select candidate markers, which were validated by immunohistochemistry. Three proteins were identified in the training set and validated in the test set by Kaplan-Meier method and immunohistochemistry (IHC): TrpRS as a good prognostic markers and DP and TSP1 as bad prognostic markers. We propose the establishment of an IHC test to calculate the score of TrpRS, DP, and TSP1 in TNBC tumors to evaluate the degree of aggressiveness of the tumors. Finally, we propose that DP and TSP1 could provide therapeutic targets for specific treatments.

  7. Detection and quantitation of infectious pancreatic necrosis virus by real-time reverse transcriptase-polymerase chain reaction using lethal and non-lethal tissue sampling.

    PubMed

    Bowers, Robert M; Lapatra, Scott E; Dhar, Arun K

    2008-02-01

    Infectious pancreatic necrosis virus (IPNV) is a bisegmented double-stranded RNA virus belonging to the family Birnaviridae, genus Aquabirnavirus, which is a major viral pathogen of salmonid fish. The virus infects wild and cultured salmonids, causing high mortality in juvenile trout and salmon. A highly sensitive and specific real-time RT-PCR assay using the fluorogenic dye SYBR((R)) Green I was developed for the detection and quantitation of IPNV in rainbow trout (Oncorhynchus mykiss). Rainbow trout were infected experimentally with IPNV in the laboratory by injection or immersion and then pectoral fin, spleen, and head kidney samples were collected for analysis. The corresponding cDNA was synthesized using DNase I-treated total RNA and then real-time RT-PCR was performed using primers based on the IPNV non-structural protein gene, designated as either NS or VP4. Rainbow trout beta-actin and elongation factor 1alpha (EF-1alpha) genes were used as internal controls. Using real-time RT-PCR, the virus was successfully detected in pectoral fin, spleen, and head kidney tissue samples. The dissociation curves for each amplicon showed a single melting peak at 83, 81.5, and 84 degrees C for IPNV NS, trout beta-actin, and EF-1alpha genes, respectively. The amplicon size and nucleotide sequence was used to confirm the specificity of the products. Using a dilution series of in vitro transcribed RNA, IPNV was reliably detected down to 10 RNA copies and had a dynamic range up to 10(7) RNA copies. A time course assay, using immersion challenged samples, revealed that the virus could be detected in pectoral fin, spleen, and head kidney as early as 24h post-challenge. The average viral load in all three tissues increased over time, reaching its highest level at 21 days post-challenge, which was followed by a slight decrease at 28 days post-challenge. IPNV load in pectoral fin tissue was comparable to the viral load in spleen and head kidney tissues, indicating that pectoral fin

  8. 1300 nm and 890 nm OCT images of oral cancer tissue engineered models and biopsy samples offer complimentary performance (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Boadi, Joseph; Byers, Robert A.; Fernandes, Jon; Mittar, Shweta; Hearnden, Vanessa; Lu, Zenghai; MacNeil, Sheila; Thornhill, Martin; Murdoch, Craig; Hunter, Keith D.; McKechnie, Alasdair; Matcher, Stephen J.

    2016-02-01

    OCT has demonstrated great potential to non-invasively detect oral epithelial cancers, potentially guiding biopsy and surgical resection. On non-ophthalmic tissues the preferred illumination wavelength is 1300 nm. Previous studies on skin have shown that useful image data can also be obtained at shorter wavelengths, with systems at 1060 nm and 820 nm offering reduced depth penetration but higher contrast. Here we apply a similar comparison to tissue engineered models of oral cancer and also to human biopsy samples, generally finding a similar trend. 1300 nm multi-beam OCT (Michelson Diagnostics EX1301) visualises stromal structures and surface keratin more clearly, providing useful image contrast down to around 1 mm. This system was compared with an ultra-high resolution home-built system operating at 890 nm (2.5 micron resolution vs 7.5 micron axial resolution for the EX1301). The UHR system reveals epithelial features more clearly, especially in the DOK pre-invasive cell line model and the biopsy samples. The relative effects of center wavelength vs axial resolution in generating the differential, wavelength-dependent contrast are assessed and the OCT biopsy images are compared with contemporary histology.

  9. Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples.

    PubMed

    Barcelos, Denise; Franco, Marcello F; Leão, Sylvia Cardoso

    2008-01-01

    Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10% non-buffered or 10% buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10% non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.

  10. Postmortem interval alters the water relaxation and diffusion properties of rat nervous tissue--implications for MRI studies of human autopsy samples.

    PubMed

    Shepherd, Timothy M; Flint, Jeremy J; Thelwall, Peter E; Stanisz, Greg J; Mareci, Thomas H; Yachnis, Anthony T; Blackband, Stephen J

    2009-02-01

    High-resolution imaging of human autopsy tissues may improve our understanding of in vivo MRI findings, but interpretation is complicated because samples are obtained by immersion fixation following a postmortem interval (PMI). This study tested the hypotheses that immersion fixation and PMI's from 0-24 h would alter the water relaxation and diffusion properties in rat cortical slice and spinal cord models of human nervous tissue. Diffusion data collected from rat cortical slices at multiple diffusion times (10-60 ms) and b-values (7-15,000 s/mm(2)) were analyzed using a two-compartment model with exchange. Rat spinal cords were characterized with standard diffusion tensor imaging (21 directions, b=1250 s/mm(2)). Switching from perfusion- to immersion-fixation at 0 h PMI altered most MRI properties of rat cortical slices and spinal cords, including a 22% decrease in fractional anisotropy (P<0.001). After 4 h PMI, cortical slice T(1) and T(2) increased 22% and 65% respectively (P<0.001), transmembrane water exchange decreased 23% (P<0.001) and intracellular proton fraction increased 25% (P=0.002). After 6 h PMI, spinal cord white matter fractional anisotropy had decreased 38% (P<0.001). MRI property changes were observed for PMIs up to 24 h. The MRI changes correlated with protease activity and histopathological signs of autolysis. Thus, immersion fixation and/or even short PMIs (4-6 h) altered the MRI properties of rat nervous tissue. This suggests comparisons between in vivo clinical MRI and MRI data from human autopsy tissues should be interpreted with caution.

  11. Determination of medicinal and illicit drugs in post mortem dental hard tissues and comparison with analytical results for body fluids and hair samples.

    PubMed

    Klima, Miriam; Altenburger, Markus J; Kempf, Jürgen; Auwärter, Volker; Neukamm, Merja A

    2016-08-01

    In burnt or skeletonized bodies dental hard tissue sometimes is the only remaining specimen available. Therefore, it could be used as an alternative matrix in post mortem toxicology. Additionally, analysis of dental tissues could provide a unique retrospective window of detection. For forensic interpretation, routes and rates of incorporation of different drugs as well as physicochemical differences between tooth root, tooth crown and carious material have to be taken into account. In a pilot study, one post mortem tooth each from three drug users was analyzed for medicinal and illicit drugs. The pulp was removed in two cases; in one case the tooth was root canal treated. The teeth were separated into root, crown and carious material and drugs were extracted from the powdered material with methanol under ultrasonication. The extracts were screened for drugs by LC-MS(n) (ToxTyper™) and quantitatively analyzed with LC-ESI-MS/MS in MRM mode. The findings were compared to the analytical results for cardiac blood, femoral blood, urine, stomach content and hair. In dental hard tissues, 11 drugs (amphetamine, MDMA, morphine, codeine, norcodeine, methadone, EDDP, fentanyl, tramadol, diazepam, nordazepam, and promethazine) could be detected and concentrations ranged from approximately 0.13pg/mg to 2,400pg/mg. The concentrations declined in the following order: carious material>root>crown. Only the root canal treated tooth showed higher concentrations in the crown than in the root. In post mortem toxicology, dental hard tissue could be a useful alternative matrix facilitating a more differentiated consideration of drug consumption patterns, as the window of detection seems to overlap those for body fluids and hair.

  12. Analysis of sequences from field samples reveals the presence of the recently described pepper vein yellows virus (genus Polerovirus) in six additional countries.

    PubMed

    Knierim, Dennis; Tsai, Wen-Shi; Kenyon, Lawrence

    2013-06-01

    Polerovirus infection was detected by reverse transcription polymerase chain reaction (RT-PCR) in 29 pepper plants (Capsicum spp.) and one black nightshade plant (Solanum nigrum) sample collected from fields in India, Indonesia, Mali, Philippines, Thailand and Taiwan. At least two representative samples for each country were selected to generate a general polerovirus RT-PCR product of 1.4 kb length for sequencing. Sequence analysis of the partial genome sequences revealed the presence of pepper vein yellows virus (PeVYV) in all 13 samples. A 1990 Australian herbarium sample of pepper described by serological means as infected with capsicum yellows virus (CYV) was identified by sequence analysis of a partial CP sequence as probably infected with a potato leaf roll virus (PLRV) isolate.

  13. The limitations of tissue-oxygen measurement and positron emission tomography as additional methods for postoperative breast reconstruction free-flap monitoring.

    PubMed

    Schrey, Aleksi; Niemi, Tarja; Kinnunen, Ilpo; Minn, Heikki; Vahlberg, Tero; Kalliokoski, Kari; Suominen, Erkki; Grénman, Reidar; Aitasalo, Kalle

    2010-02-01

    Twelve patients who underwent breast reconstruction with a microvascular flap were monitored postoperatively with continuous partial tissue oxygenation (p(ti)O(2)) measurement. The regional blood flow (BF) of the entire flap was evaluated with positron emission tomography (PET) using oxygen-15-labelled water on the first postoperative (POP) morning to achieve data of the perfusion of the entire flap. A re-exploration was carried out if the p(ti)O(2) value remained lower than 15 mmHg for over 30 min. The mean p(ti)O(2) value of the flaps was 52.9+/-5.5 mmHg, whereas the mean BF values were 3.3+/-1.0 ml per 100 g min(-1). One false-positive result was detected by p(ti)O(2) measurement, resulting in an unnecessary re-exploration. Another re-operation suggested by the low p(ti)O(2) results was avoided due to the normal BF results assessed with PET. Totally, three flaps were re-explored. This prospective study suggests that continuous tissue-oxygen measurement with a polarographic needle probe is reliable for monitoring free breast flaps from one part of the flap, but assessing perfusion of the entire flap requires more complex monitoring methods, for example, PET. Clinical examination by experienced personnel remains important in free-breast-flap monitoring. PET could be useful in assessing free-flap perfusion in selected high-risk patients as an alternative to a re-operation when clinical examination and evaluation by other means are unreliable or present controversial results.

  14. FT-Raman and chemometric tools for rapid determination of quality parameters in milk powder: Classification of samples for the presence of lactose and fraud detection by addition of maltodextrin.

    PubMed

    Rodrigues Júnior, Paulo Henrique; de Sá Oliveira, Kamila; de Almeida, Carlos Eduardo Rocha; De Oliveira, Luiz Fernando Cappa; Stephani, Rodrigo; Pinto, Michele da Silva; de Carvalho, Antônio Fernandes; Perrone, Ítalo Tuler

    2016-04-01

    FT-Raman spectroscopy has been explored as a quick screening method to evaluate the presence of lactose and identify milk powder samples adulterated with maltodextrin (2.5-50% w/w). Raman measurements can easily differentiate samples of milk powder, without the need for sample preparation, while traditional quality control methods, including high performance liquid chromatography, are cumbersome and slow. FT-Raman spectra were obtained from samples of whole lactose and low-lactose milk powder, both without and with addition of maltodextrin. Differences were observed between the spectra involved in identifying samples with low lactose content, as well as adulterated samples. Exploratory data analysis using Raman spectroscopy and multivariate analysis was also developed to classify samples with PCA and PLS-DA. The PLS-DA models obtained allowed to correctly classify all samples. These results demonstrate the utility of FT-Raman spectroscopy in combination with chemometrics to infer about the quality of milk powder.

  15. Real-time optical coherence tomography observation of retinal tissue damage during laser photocoagulation therapy on ex-vivo porcine samples

    NASA Astrophysics Data System (ADS)

    Steiner, P.; Považay, B.; Stoller, M.; Morgenthaler, P.; Inniger, D.; Arnold, P.; Sznitman, R.; Meier, Ch.

    2015-07-01

    Retinal laser photocoagulation represents a widely used treatment for retinal pathologies such as diabetic chorioretinopathy or diabetic edema. For effective treatment, an appropriate choice of the treatment energy dose is crucial to prevent excessive tissue damage caused by over-irradiation of the retina. In this manuscript we investigate simultaneous and time-resolved optical coherence tomography for its applicability to provide feedback to the ophthalmologist about the introduced retinal damage during laser photocoagulation. Time-resolved and volumetric optical coherence tomography data of 96 lesions on ex-vivo porcine samples, set with a 577 nm laser prototype and irradiance of between 300 and 8800 W=cm2 were analyzed. Time-resolved scans were compared to volumetric scans of the lesion and correlated with ophthalmoscopic visibility. Lastly, image parameters extracted from optical coherence tomography Mscans, suitable for lesion classification were identified. Results presented in this work support the hypothesis that simultaneous optical coherence tomography provides valuable information about the extent of retinal tissue damage and may be used to guide retinal laser photocoagulation in the future.

  16. Rapid identification and characterization of Penicillium marneffei using multiplex ligation-dependent probe amplification (MLPA) in paraffin-embedded tissue samples.

    PubMed

    Zhang, Jun-Min; Sun, Jiu-Feng; Feng, Pei-Ying; Li, Xi-Qing; Lu, Chang-Ming; Lu, Sha; Cai, Wen-Ying; Xi, Li-Yan; de Hoog, G S

    2011-04-01

    Penicillium marneffei infection is a deadly disease and early diagnosis leads to prompt and appropriate antifungal therapy. To develop a sensitive method to diagnose P. marneffei infection, a multiplex ligation-dependent probe amplification (MLPA) assay was adapted. This method can rapidly and specifically detect P. marneffei DNA in cultured cells and paraffin-embedded tissue samples. Three pairs of probes were designed for amplifying the internally (intergenic) transcribed spacer (ITS) region of P. marneffei rRNA using a systematic phylogenetic analysis. These three probe sets produced three amplicons of 198, 166, and 152 bp, respectively, specific for P. marneffei. In contrast, there was only one 198 bp amplicon produced for Talaromyces stipitatus, and one 152 bp amplicon for P. funiculosum, T. intermedius and T. derxii. The probes did not amplify any other reference strains. An array of 40 P. marneffei strains isolated from human patients, bamboo rat, and the local environment was tested by using MLPA, and all were positively identified. Most importantly, P. marneffei in paraffin-embedded tissue specimens from infected human patients was positively amplified by MLPA. The sensitivity and specificity of the MLPA assay could be a useful tool for prompt diagnosis, pathogen characterization, and epidemiological studies of fungal infections.

  17. Promoter region hypermethylation and mRNA expression of MGMT and p16 genes in tissue and blood samples of human premalignant oral lesions and oral squamous cell carcinoma.

    PubMed

    Bhatia, Vikram; Goel, Madhu Mati; Makker, Annu; Tewari, Shikha; Yadu, Alka; Shilpi, Priyanka; Kumar, Sandeep; Agarwal, S P; Goel, Sudhir K

    2014-01-01

    Promoter methylation and relative gene expression of O(6)-methyguanine-DNA-methyltransferase (MGMT) and p16 genes were examined in tissue and blood samples of patients with premalignant oral lesions (PMOLs) and oral squamous cell carcinoma (OSCC). Methylation-specific PCR and reverse transcriptase PCR were performed in 146 tissue and blood samples from controls and patients with PMOLs and OSCC. In PMOL group, significant promoter methylation of MGMT and p16 genes was observed in 59% (P = 0.0010) and 57% (P = 0.0016) of tissue samples, respectively, and 39% (P = 0.0135) and 33% (P = 0.0074) of blood samples, respectively. Promoter methylation of both genes was more frequent in patients with OSCC, that is, 76% (P = 0.0001) and 82% (P = 0.0001) in tissue and 57% (P = 0.0002) and 70% (P = 0.0001) in blood, respectively. Significant downregulation of MGMT and p16 mRNA expression was observed in both tissue and blood samples from patients with PMOLs and OSCC. Hypermethylation-induced transcriptional silencing of MGMT and p16 genes in both precancer and cancer suggests important role of these changes in progression of premalignant state to malignancy. Results support use of blood as potential surrogate to tissue samples for screening or diagnosing PMOLs and early OSCC.

  18. Promoter Region Hypermethylation and mRNA Expression of MGMT and p16 Genes in Tissue and Blood Samples of Human Premalignant Oral Lesions and Oral Squamous Cell Carcinoma

    PubMed Central

    Bhatia, Vikram; Makker, Annu; Tewari, Shikha; Yadu, Alka; Shilpi, Priyanka; Kumar, Sandeep; Agarwal, S. P.; Goel, Sudhir K.

    2014-01-01

    Promoter methylation and relative gene expression of O6-methyguanine-DNA-methyltransferase (MGMT) and p16 genes were examined in tissue and blood samples of patients with premalignant oral lesions (PMOLs) and oral squamous cell carcinoma (OSCC). Methylation-specific PCR and reverse transcriptase PCR were performed in 146 tissue and blood samples from controls and patients with PMOLs and OSCC. In PMOL group, significant promoter methylation of MGMT and p16 genes was observed in 59% (P = 0.0010) and 57% (P = 0.0016) of tissue samples, respectively, and 39% (P = 0.0135) and 33% (P = 0.0074) of blood samples, respectively. Promoter methylation of both genes was more frequent in patients with OSCC, that is, 76% (P = 0.0001) and 82% (P = 0.0001) in tissue and 57% (P = 0.0002) and 70% (P = 0.0001) in blood, respectively. Significant downregulation of MGMT and p16 mRNA expression was observed in both tissue and blood samples from patients with PMOLs and OSCC. Hypermethylation-induced transcriptional silencing of MGMT and p16 genes in both precancer and cancer suggests important role of these changes in progression of premalignant state to malignancy. Results support use of blood as potential surrogate to tissue samples for screening or diagnosing PMOLs and early OSCC. PMID:24991542

  19. Use of a novel radiometric method to assess the inhibitory effect of donepezil on acetylcholinesterase activity in minimally diluted tissue samples

    PubMed Central

    Kikuchi, Tatsuya; Okamura, Toshimitsu; Arai, Takuya; Obata, Takayuki; Fukushi, Kiyoshi; Irie, Toshiaki; Shiraishi, Tetsuya

    2010-01-01

    Background and purpose: Cholinesterase inhibitors have been widely used for the treatment of patients with dementia. Monitoring of the cholinesterase activity in the blood is used as an indicator of the effect of the cholinesterase inhibitors in the brain. The selective measurement of cholinesterase with low tissue dilution is preferred for accurate monitoring; however, the methods have not been established. Here, we investigated the effect of tissue dilution on the action of cholinesterase inhibitors using a novel radiometric method with selective substrates, N-[14C]methylpiperidin-4-yl acetate ([14C]MP4A) and (R)-N-[14C]methylpiperidin-3-yl butyrate ([14C]MP3B_R), for AChE and butyrylcholinesterase (BChE) respectively. Experimental approach: We investigated the kinetics of hydrolysis of [14C]-MP4A and [14C]-MP3B_R by cholinesterases, and evaluated the selectivity of [14C]MP4A and [14C]MP3B_R for human AChE and BChE, respectively, compared with traditional substrates. Then, IC50 values of cholinesterase inhibitors in minimally diluted and highly diluted tissues were measured with [14C]MP4A and [14C]MP3B_R. Key results: AChE and BChE activities were selectively measured as the first-order hydrolysis rates of [14C]-MP4A and [14C]MP3B_R respectively. The AChE selectivity of [14C]MP4A was an order of magnitude higher than traditional substrates used for the AChE assay. The IC50 values of specific AChE and BChE inhibitors, donepezil and ethopropazine, in 1.2-fold diluted human whole blood were much higher than those in 120-fold diluted blood. In addition, the IC50 values of donepezil in monkey brain were dramatically decreased as the tissue was diluted. Conclusions and implications: This method would effectively monitor the activity of cholinesterase inhibitors used for therapeutics, pesticides and chemical warfare agents. PMID:20401964

  20. Maps showing mines, quarries, oil and gas activity, and sample localities in and near the Sipsey Wilderness and additions, Lawrence and Winston Counties, Alabama

    SciTech Connect

    Mory, P.C.; Behum, P.T.; Ross, R.B. Jr.

    1982-01-01

    This report presents the results of a mineral survey of the Sipsey Wilderness and additions, William B. Bankhead National Forest, Lawrence and Winston Counties, Alabama. The survey includes: limestone quarrying, coal mining, and oil and gas activity. 7 references, 5 figures, 2 tables.

  1. Capillary electrophoretic separation of humic substances using hydroxyethyl cellulose as a buffer additive and its application to characterization of humic substances in a river water sample.

    PubMed

    Takahashi, Toru; Kawana, Jun; Hoshino, Hitoshi

    2009-01-01

    We have developed a concise tool for the investigation of the transition of humic substances in environmental water. The separation of water-soluble humic substances was achieved rapidly and effectively by capillary electrophoresis using a polyacrylamide-coated capillary and a phosphate electrophoretic buffer solution (pH 7.0) containing hydroxyethyl cellulose. The separation mechanism was assessed using the ultrafiltration technique. The effect of the complexation of humic substances with metal ions was studied by using the proposed method. When Fe(III) ions or EDTA was added to the sample solution of fulvic acid, a distinct change in the electropherogram pattern based on the conformational change of fulvic acid was observed. The successful application of the proposed method to the characterization of humic substances in a river water sample was also demonstrated.

  2. Molecular characterization of Staphylococcus aureus isolates from skin and soft tissue infections samples and healthy carriers in the Central Slovenia region.

    PubMed

    Svent-Kucina, Natasa; Pirs, Mateja; Kofol, Romina; Blagus, Rok; Smrke, Dragica Maja; Bilban, Marjan; Seme, Katja

    2016-04-01

    Staphylococcus aureus is among the most important human pathogens. It is associated with different infections and is a major cause of skin and soft tissue infections (SSTIs). The aim of our study was to compare S. aureus isolates associated with SSTIs with isolates obtained from healthy carriers in the Central Slovenia region in terms of antimicrobial susceptibility, genetic diversity by clonal complex (CC)/sequence type, spa type, and by toxin gene profiling. In total, 274 S. aureus isolates were collected prospectively by culturing wound samples from 461 SSTI patients and nasal samples from 451 healthy carriers. We have demonstrated high heterogeneity in terms of CCs and spa type in both groups of isolates. The main clone among SSTI strains was Panton-Valentine leukocidin gene (pvl) positive CC121, whereas the main clone among carrier strains was CC45 carrying a large range of toxin genes. The main spa type in both groups was t091. Pvl was more frequently present in SSTI strains (31.2% SSTI vs 3.6% carrier strains) and staphylococcal enterotoxin C was more frequently present in carrier strains (1.6% SSTI vs 17.0% carrier strains). We have also demonstrated that methicillin-resistant S. aureus was a rare cause (2.8%) of SSTIs in our region.

  3. Endoscopic ultrasound-guided tissue sampling of small subepithelial tumors of the upper gastrointestinal tract with a 22-gauge core biopsy needle

    PubMed Central

    Schlag, Christoph; Menzel, Christoph; Götzberger, Manuela; Nennstiel, Simon; Klare, Peter; Wagenpfeil, Stefan; Schmid, Roland M.; Weirich, Gregor; von Delius, Stefan

    2017-01-01

    Background and study aims The optimal approach to small subepithelial tumors (SETs) of the upper gastrointestinal tract remains inconclusive. The aim of this study was to evaluate endoscopic ultrasound-guided fine needle biopsy (EUS-FNB) for less invasive tissue sampling of small SETs of the upper gastrointestinal tract. Patients and methods In this prospective observational study patients with small ( ≤ 3 cm) SETs of the upper gastrointestinal tract were eligible and underwent EUS-FNB with a 22-gauge core biopsy needle. The main outcome measure was the diagnostic yield. The number of obtained core biopsies was also assessed. Results Twenty patients were included. The mean SET size was 16 mm (range 10 – 27 mm). EUS-FNB was technically feasible in all cases and no complications were observed. The diagnostic yield was 75 %. Core biopsy specimens were obtained in only 25 % of cases. Conclusion EUS-FNB with a 22-gauge core biopsy needle of small SETs can achieve a definite diagnosis in the majority of cases. However, because core samples cannot regularly be obtained, EUS-FNB seems not to be convincingly superior to standard EUS-FNA in this setting PMID:28299351

  4. Assessment of heavy metals in water samples and tissues of edible fish species from Awassa and Koka Rift Valley Lakes, Ethiopia.

    PubMed

    Dsikowitzky, Larissa; Mengesha, Mesfin; Dadebo, Elias; de Carvalho, Carlos Eduardo Veiga; Sindern, Sven

    2013-04-01

    The Ethiopian Rift Valley Lakes host populations of edible fish species including Oreochromis niloticus, Labeobarbus intermedius and Clarias gariepinus, which are harvested also in other tropical countries. We investigated the occurrence of six heavy metals in tissues of these fish species as well as in the waters of Lake Koka and Lake Awassa. Both lakes are affected by industrial effluents in their catchments, making them ideal study sites. Mercury concentrations were very low in the water samples, but concentrations in the fish samples were relatively high, suggesting a particularly high bioaccumulation tendency as compared with the other investigated metals. Mercury was preferentially accumulated in the fish liver or muscle. It was the only metal with species-specific accumulation with highest levels found in the predatory species L. intermedius. Lower mercury concentrations in O. niloticus could be attributed to the lower trophic level, whereas mercury values in the predatory C. gariepinus were unexpectedly low. This probably relates to the high growth rate of this species resulting in biodilution of mercury. Accumulation of lead, selenium, chromium, arsenic and cadmium did not differ between species, indicating that these elements are not biomagnified in the food chain. Values of cadmium, selenium and arsenic were highest in fish livers, while lead and chromium levels were highest in the gills, which could be related to the uptake pathway. A significant impact of the industrial discharges on the occurrence of metals in the lakes could not be detected, and the respective concentrations in fish do not pose a public health hazard.

  5. Effects of Addition of Linseed and Marine Algae to the Diet on Adipose Tissue Development, Fatty Acid Profile, Lipogenic Gene Expression, and Meat Quality in Lambs

    PubMed Central

    Urrutia, Olaia; Mendizabal, José Antonio; Insausti, Kizkitza; Soret, Beatriz; Purroy, Antonio; Arana, Ana

    2016-01-01

    This study examined the effect of linseed and algae on growth and carcass parameters, adipocyte cellularity, fatty acid profile and meat quality and gene expression in subcutaneous and intramuscular adipose tissues (AT) in lambs. After weaning, 33 lambs were fed three diets up to 26.7 ± 0.3 kg: Control diet (barley and soybean); L diet (barley, soybean and 10% linseed) and L-A diet (barley, soybean, 5% linseed and 3.89% algae). Lambs fed L-A diet showed lower average daily gain and greater slaughter age compared to Control and L (P < 0.001). Carcass traits were not affected by L and L-A diets, but a trend towards greater adipocyte diameter was observed in L and L-A in the subcutaneous AT (P = 0.057). Adding either linseed or linseed and algae increased α-linolenic acid and eicosapentaenoic acid contents in both AT (P < 0.001); however, docosahexaenoic acid was increased by L-A (P < 0.001). The n-6/n-3 ratio decreased in L and L-A (P < 0.001). Algae had adverse effects on meat quality, with greater lipid oxidation and reduced ratings for odor and flavor. The expression of lipogenic genes was downregulated in the subcutaneous AT (P < 0.05): acetyl-CoA carboxylase 1 (ACACA) in L and L-A and lipoprotein lipase (LPL) and stearoyl-CoA desaturase (SCD) in L-A. Fatty acid desaturase 1 (FADS1), fatty acid desaturase 2 (FADS2) and fatty acid elongase 5 (ELOVL5) were unaffected. In the subcutaneous AT, supplementing either L or L-A increased peroxisome proliferator-activated receptor gamma (PPARG) and CAAT-enhancer binding protein alpha (CEBPA) (P < 0.05), although it had no effect on sterol regulatory element-binding factor 1 (SREBF1). In the intramuscular AT, expression of ACACA, SCD, FADS1 and FADS2 decreased in L and L-A (P < 0.001) and LPL in L (P < 0.01), but PPARG, CEBPA and SREBF1 were unaffected. PMID:27253325

  6. Phase II Trial of Cetuximab and Conformal Radiotherapy Only in Locally Advanced Pancreatic Cancer with Concurrent Tissue Sampling Feasibility Study1

    PubMed Central

    Rembielak, Agata I; Jain, Pooja; Jackson, Andrew S; Green, Melanie M; Santorelli, Gillian R; Whitfield, Gillian A; Crellin, Adrian; Garcia-Alonso, Angel; Radhakrishna, Ganesh; Cullen, James; Taylor, M Ben; Swindell, Ric; West, Catharine M; Valle, Juan; Saleem, Azeem; Price, Patricia M

    2014-01-01

    BACKGROUND: Preclinical data have indicated the anti-epidermal growth factor receptor (EGFR) agent cetuximab (Erbitux) as a radiosensitizer in pancreatic cancer, but this has not been specifically addressed in a clinical study. We report the results of an original study initiated in 2007, where cetuximab was tested with radiotherapy (RT) alone in locally advanced pancreatic cancer in a phase II trial (PACER). METHODS: Patients (n = 21) received cetuximab loading dose (400 mg/m2) and weekly dose (250 mg/m2) during RT (50.4 Gy in 28 fractions). Toxicity and disease response end point data were prospectively assessed. A feasibility study of on-trial patient blood and skin sampling was incorporated. RESULTS: Treatment was well tolerated, and toxicity was low; most patients (71%) experienced acute toxicities of grade 2 or less. Six months posttreatment, stable local disease was achieved in 90% of evaluable patients, but only 33% were free from metastatic progression. Median overall survival was 7.5 months, and actuarial survival was 33% at 1 year and 11% at 3 years, reflecting swift metastatic progression in some patients but good long-term control of localized disease in others. High-grade acneiform rash (P = .0027), posttreatment stable disease (P = .0059), and pretreatment cancer antigen 19.9 (CA19.9) level (P = .0042) associated with extended survival. Patient skin and blood samples yielded sufficient RNA and good quality protein, respectively. CONCLUSIONS: The results indicate that cetuximab inhibits EGFR-mediated radioresistance to achieve excellent local control with minimal toxicity but does not sufficiently control metastatic progression in all patients. Translational studies of patient tissue samples may yield molecular information that may enable individual treatment response prediction. PMID:24772208

  7. Additional notes on clinical repeated-dose pharmacokinetic trials applying a peak-and-trough sampling design to estimate oral clearance.

    PubMed

    Takaai, Mari; Kayano, Yuichiro; Shimizu, Takako; Taguchi, Masato; Hashimoto, Yukiya

    2008-01-01

    In the previous study, we performed a simulation of a clinical pharmacokinetic trial, in which blood was sampled at two time points corresponding to the peak concentration (C(peak)) and trough concentration (C(trough)) following repetitive oral administration at the dose, D, and dosing interval, tau. The approximate oral clearance (CL/F(approx)), estimated as 2 x D/(C(peak) x tau+C(trough) x tau), is accurate for drugs with an elimination half-life comparative to or longer than tau; however, it was suggested that we might not use CL/F(approx) for drugs with a considerably short elimination half-life relative to tau. In the present study, we evaluated the accuracy of the alternative oral clearance (CL/F(exp)) estimated by the simple monoexponential model. In contrast to CL/F(approx), CL/F(exp) was accurate for drugs with a short elimination half-life relative to tau. The present finding in conjunction with our previous study suggested that the peak-and-trough sampling design is promising for the clinical repeated-dose pharmacokinetic trial for drugs with not only slow but also rapid elimination from the body. We think that the accuracy and precision of the two analysis methods to estimate oral clearance (CL/F(approx) and CL/F(exp)) for a target drug should be evaluated carefully before and after a real clinical trial.

  8. Evaluation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in Pyrus pyrifolia using different tissue samples and seasonal conditions.

    PubMed

    Imai, Tsuyoshi; Ubi, Benjamin E; Saito, Takanori; Moriguchi, Takaya

    2014-01-01

    We have evaluated suitable reference genes for real time (RT)-quantitative PCR (qPCR) analysis in Japanese pear (Pyrus pyrifolia). We tested most frequently used genes in the literature such as β-Tubulin, Histone H3, Actin, Elongation factor-1α, Glyceraldehyde-3-phosphate dehydrogenase, together with newly added genes Annexin, SAND and TIP41. A total of 17 primer combinations for these eight genes were evaluated using cDNAs synthesized from 16 tissue samples from four groups, namely: flower bud, flower organ, fruit flesh and fruit skin. Gene expression stabilities were analyzed using geNorm and NormFinder software packages or by ΔCt method. geNorm analysis indicated three best performing genes as being sufficient for reliable normalization of RT-qPCR data. Suitable reference genes were different among sample groups, suggesting the importance of validation of gene expression stability of reference genes in the samples of interest. Ranking of stability was basically similar between geNorm and NormFinder, suggesting usefulness of these programs based on different algorithms. ΔCt method suggested somewhat different results in some groups such as flower organ or fruit skin; though the overall results were in good correlation with geNorm or NormFinder. Gene expression of two cold-inducible genes PpCBF2 and PpCBF4 were quantified using the three most and the three least stable reference genes suggested by geNorm. Although normalized quantities were different between them, the relative quantities within a group of samples were similar even when the least stable reference genes were used. Our data suggested that using the geometric mean value of three reference genes for normalization is quite a reliable approach to evaluating gene expression by RT-qPCR. We propose that the initial evaluation of gene expression stability by ΔCt method, and subsequent evaluation by geNorm or NormFinder for limited number of superior gene candidates will be a practical way of finding out

  9. Processes and procedures for a worldwide biological samples distribution; product assurance and logistic activities to support the mice drawer system tissue sharing event

    NASA Astrophysics Data System (ADS)

    Benassai, Mario; Cotronei, Vittorio

    The Mice Drawer System (MDS) is a scientific payload developed by the Italian Space Agency (ASI), it hosted 6 mice on the International Space Station (ISS) and re-entered on ground on November 28, 2009 with the STS 129 at KSC. Linked to the MDS experiment, a Tissue Sharing Program (TSP), was developed in order to make available to 16 Payload Investigators (PI) (located in USA, Canada, EU -Italy, Belgium and Germany -and Japan) the biological samples coming from the mice. ALTEC SpA (a PPP owned by ASI, TAS-I and local institutions) was responsible to support the logistics aspects of the MDS samples for the first MDS mission, in the frame of Italian Space Agency (ASI) OSMA program (OSteoporosis and Muscle Atrophy). The TSP resulted in a complex scenario, as ASI, progressively, extended the original OSMA Team also to researchers from other ASI programs and from other Agencies (ESA, NASA, JAXA). The science coordination was performed by the University of Genova (UNIGE). ALTEC has managed all the logistic process with the support of a specialized freight forwarder agent during the whole shipping operation phases. ALTEC formalized all the steps from the handover of samples by the dissection Team to the packaging and shipping process in a dedicated procedure. ALTEC approached all the work in a structured way, performing: A study of the aspects connected to international shipments of biological samples. A coopera-tive work with UNIGE/ASI /PIs to identify all the needs of the various researchers and their compatibility. A complete revision and integration of shipment requirements (addresses, tem-peratures, samples, materials and so on). A complete definition of the final shipment scenario in terms of boxes, content, refrigerant and requirements. A formal approach to identification and selection of the most suited and specialized Freight Forwarder. A clear identification of all the processes from sample dissection by PI Team, sample processing, freezing, tube preparation

  10. Rapid optimized separation of bromide in serum samples with capillary zone electrophoresis by using glycerol as additive to the background electrolyte.

    PubMed

    Pascali, Jennifer P; Liotta, Eloisa; Gottardo, Rossella; Bortolotti, Federica; Tagliaro, Franco

    2009-04-10

    After decades of neglect, bromide has recently been re-introduced in therapy as an effective anti-epileptic drug. The present paper describes the methodological optimization and validation of a method based on capillary zone electrophoresis for the rapid determination of bromide in serum using a high-viscosity buffer and a short capillary (10 cm). The optimized running buffer was composed of 90 mM sodium tetraborate, 10mM sodium chloride, pH 9.24 and 25% glycerol. The separation was carried out at 25 kV at a temperature of 20 degrees C. Detection was by direct UV absorption at 200 nm wavelength. The limit of detection (signal-to-noise ratio=5) in serum was 0.017 mM. The precision of the method was verified in blank serum samples spiked with bromide, obtaining intra-day and day-to-day tests, relative standard deviation values

  11. Oral pathology follow-up by means of micro-Raman spectroscopy on tissue and blood serum samples: an application of wavelet and multivariate data analysis

    NASA Astrophysics Data System (ADS)

    Delfino, I.; Camerlingo, C.; Zenone, F.; Perna, G.; Capozzi, V.; Cirillo, N.; Gaeta, G. M.; De Mol, E.; Lepore, M.

    2009-02-01

    Pemphigus vulgaris (PV) is a potentially fatal autoimmune disease that cause blistering of the skin and oral cavity. It is characterized by disruption of cell-cell adhesion within the suprabasal layers of epithelium, a phenomenon termed acantholysis Patients with PV develop IgG autoantibodies against normal constituents of the intercellular substance of keratinocytes. The mechanisms by which such autoantibodies induce blisters are not clearly understood. The qualitative analysis of such effects provides important clues in the search for a specific diagnosis, and the quantitative analysis of biochemical abnormalities is important in measuring the extent of the disease process, designing therapy and evaluating the efficacy of treatment. Improved diagnostic techniques could permit the recognition of more subtle forms of disease and reveal incipient lesions clinically unapparent, so that progression of potentially severe forms could be reversed with appropriate treatment. In this paper, we report the results of our micro-Raman spectroscopy study on tissue and blood serum samples from ill, recovered and under therapy PV patients. The complexity of the differences among their characteristic Raman spectra has required a specific strategy to obtain reliable information on the illness stage of the patients For this purpose, wavelet techniques and advanced multivariate analysis methods have been developed and applied to the experimental Raman spectra. Promising results have been obtained.

  12. Pathology in Continuous Infusion Studies in Rodents and Non-Rodents and ITO (Infusion Technology Organisation)-Recommended Protocol for Tissue Sampling and Terminology for Procedure-Related Lesions

    PubMed Central

    Weber, Klaus; Mowat, Vasanthi; Hartmann, Elke; Razinger, Tanja; Chevalier, Hans-Jörg; Blumbach, Kai; Green, Owen P.; Kaiser, Stefan; Corney, Stephen; Jackson, Ailsa; Casadesus, Agustin

    2011-01-01

    Many variables may affect the outcome of continuous infusion studies. The results largely depend on the experience of the laboratory performing these studies, the technical equipment used, the choice of blood vessels and hence the surgical technique as well the quality of pathological evaluation. The latter is of major interest due to the fact that the pathologist is not involved until necropsy in most cases, i.e. not dealing with the complicated surgical or in-life procedures of this study type. The technique of tissue sampling during necropsy and the histology processing procedures may influence the tissues presented for evaluation, hence the pathologist may be a source of misinterpretation. Therefore, ITO proposes a tissue sampling procedure and a standard nomenclature for pathological lesions for all sites and tissues in contact with the port-access and/or catheter system. PMID:22272050

  13. Food additives

    PubMed Central

    Spencer, Michael

    1974-01-01

    Food additives are discussed from the food technology point of view. The reasons for their use are summarized: (1) to protect food from chemical and microbiological attack; (2) to even out seasonal supplies; (3) to improve their eating quality; (4) to improve their nutritional value. The various types of food additives are considered, e.g. colours, flavours, emulsifiers, bread and flour additives, preservatives, and nutritional additives. The paper concludes with consideration of those circumstances in which the use of additives is (a) justified and (b) unjustified. PMID:4467857

  14. Correlation of MLH1 and MGMT methylation levels between peripheral blood leukocytes and colorectal tissue DNA samples in colorectal cancer patients.

    PubMed

    Li, Xia; Wang, Yibaina; Zhang, Zuoming; Yao, Xiaoping; Ge, Jie; Zhao, Yashuang

    2013-11-01

    CpG island methylation in the promoter regions of the DNA mismatch repair gene mutator L homologue 1 (MLH1) and DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT) genes has been shown to occur in the leukocytes of peripheral blood and colorectal tissue. However, it is unclear whether the methylation levels in the blood leukocytes and colorectal tissue are correlated. The present study analyzed and compared the levels of MGMT and MLH1 gene methylation in the leukocytes of peripheral blood and colorectal tissues obtained from patients with colorectal cancer (CRC). The methylation levels of MGMT and MLH1 were examined using methylation-sensitive high-resolution melting (MS-HRM) analysis. A total of 44 patients with CRC were selected based on the MLH1 and MGMT gene methylation levels in the leukocytes of the peripheral blood. Corresponding colorectal tumor and normal tissues were obtained from each patient and the DNA methylation levels were determined. The correlation coefficients were evaluated using Spearman's rank test. Agreement was determined by generalized κ-statistics. Spearman's rank correlation coefficients (r) for the methylation levels of the MGMT and MLH1 genes in the leukocytes of the peripheral blood and normal colorectal tissue were 0.475 and 0.362, respectively (P=0.001 and 0.016, respectively). The agreement of the MGMT and MLH1 gene methylation levels in the leukocytes of the peripheral blood and normal colorectal tissue were graded as fair and poor (κ=0.299 and 0.126, respectively). The methylation levels of MGMT and MLH1 were moderately and weakly correlated between the patient-matched leukocytes and the normal colorectal tissue, respectively. Blood-derived DNA methylation measurements may not always represent the levels of normal colorectal tissue methylation.

  15. Analysis of glutathione levels in the brain tissue samples from HIV-1-positive individuals and subject with Alzheimer's disease and its implication in the pathophysiology of the disease process.

    PubMed

    Saing, Tommy; Lagman, Minette; Castrillon, Jeffery; Gutierrez, Eutiquio; Guilford, Frederick T; Venketaraman, Vishwanath

    2016-12-01

    HIV-1 positive individuals are at high risk for susceptibility to both pulmonary tuberculosis (TB) and extra-pulmonary TB, including TB meningitis (TBM) which is an extreme form of TB. The goals of this study are to determine the mechanisms responsible for compromised levels of glutathione (GSH) in the brain tissue samples derived from HIV-1-infected individuals and individuals with Alzheimer's disease (AD), investigate the possible underlying mechanisms responsible for GSH deficiency in these pathological conditions, and establish a link between GSH levels and pathophysiology of the disease processes. We demonstrated in the autopsied human brain tissues that the levels of total and reduced forms of GSH were significantly compromised in HIV-1 infected individuals compared to in healthy subjects and individuals with AD. Brain tissue samples derived from HIV-1-positive individuals had substantially higher levels of free radicals than that derived from healthy and AD individuals. Enzymes that are responsible for the de novo synthesis of GSH such as γ-glutamate cysteine-ligase catalytic subunit (GCLC-rate limiting step enzyme) and glutathione synthetase (GSS-enzyme involved in the second step reaction) were significantly decreased in the brain tissue samples derived from HIV-1-positive individuals with low CD4 + T-cells (< 200 cells/mm(3)) compared to healthy and AD individuals. Levels of glutathione reductase (GSR) were also decreased in the brain tissue samples derived from HIV-1 infected individuals. Overall, our findings demonstrate causes for GSH deficiency in the brain tissue from HIV-1 infected individuals explaining the possible reasons for increased susceptibility to the most severe form of extra-pulmonary TB, TBM.

  16. Simultaneous determination of methyl tert-butyl ether, its degradation products and other gasoline additives in soil samples by closed-system purge-and-trap gas chromatography-mass spectrometry.

    PubMed

    Rosell, Mònica; Lacorte, Sílvia; Barceló, Damià

    2006-11-03

    A new protocol for the simultaneous determination of methyl tert-butyl ether (MTBE); its main degradation products: tert-butyl alcohol (TBA) and tert-butyl formate (TBF); other gasoline additives, oxygenate dialkyl ethers: ethyl tert-butyl ether (ETBE), tert-amyl methyl ether (TAME) and diisopropyl ether (DIPE); aromatics: benzene, toluene, ethylbenzene and xylenes (BTEX) and other compounds causing odour events such as dicyclopentadiene (DCPD) and trichloroethylene (TCE) in soils has been developed. On the basis of US Environmental Protection Agency (EPA) method 5035A, a fully automated closed-system purge-and-trap coupled to gas chromatography/mass spectrometry (P&T-GC/MS) was optimised and permitted to detect microg/kg concentrations in solid matrices avoiding losses of volatile compounds during operation processes. Parameters optimised were the sampling procedure, sample preservation and storage, purging temperature, matrix effects and quantification mode. Using 5 g of sample, detection limits were between 0.02 and 1.63 microg/kg and acceptable method precision and accuracy was obtained provided quantification was performed using adequate internal standards. Soil samples should be analysed as soon as possible after collection, stored under -15 degrees C for not longer than 7 days if degradation products have to be analysed. The non-preservative alternative (empty vial) provided good recoveries of the most analytes when freezing the samples up to 7 day holding time, however, if biologically active soil are analysed the preservation with trisodium phosphate dodecahydrate (Na(3)PO(4).12H(2)O or TSP) is strongly recommended more than sodium bisulphate (NaHSO(4)). The method was finally applied to provide threshold and background levels of several gasoline additives in a point source and in sites not influenced by gasoline spills. The proposed method provides the directions for the future application on real samples in current monitoring programs at gasoline

  17. Effect of sample size on multi-parametric prediction of tissue outcome in acute ischemic stroke using a random forest classifier

    NASA Astrophysics Data System (ADS)

    Forkert, Nils Daniel; Fiehler, Jens

    2015-03-01

    The tissue outcome prediction in acute ischemic stroke patients is highly relevant for clinical and research purposes. It has been shown that the combined analysis of diffusion and perfusion MRI datasets using high-level machine learning techniques leads to an improved prediction of final infarction compared to single perfusion parameter thresholding. However, most high-level classifiers require a previous training and, until now, it is ambiguous how many subjects are required for this, which is the focus of this work. 23 MRI datasets of acute stroke patients with known tissue outcome were used in this work. Relative values of diffusion and perfusion parameters as well as the binary tissue outcome were extracted on a voxel-by- voxel level for all patients and used for training of a random forest classifier. The number of patients used for training set definition was iteratively and randomly reduced from using all 22 other patients to only one other patient. Thus, 22 tissue outcome predictions were generated for each patient using the trained random forest classifiers and compared to the known tissue outcome using the Dice coefficient. Overall, a logarithmic relation between the number of patients used for training set definition and tissue outcome prediction accuracy was found. Quantitatively, a mean Dice coefficient of 0.45 was found for the prediction using the training set consisting of the voxel information from only one other patient, which increases to 0.53 if using all other patients (n=22). Based on extrapolation, 50-100 patients appear to be a reasonable tradeoff between tissue outcome prediction accuracy and effort required for data acquisition and preparation.

  18. Food additives

    MedlinePlus

    ... or natural. Natural food additives include: Herbs or spices to add flavor to foods Vinegar for pickling ... Certain colors improve the appearance of foods. Many spices, as well as natural and man-made flavors, ...

  19. Development and evaluation of a reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay for detection of equine arteritis virus in equine semen and tissue samples using the POCKIT™ system.

    PubMed

    Carossino, Mariano; Lee, Pei-Yu A; Nam, Bora; Skillman, Ashley; Shuck, Kathleen M; Timoney, Peter J; Tsai, Yun-Long; Ma, Li-Juan; Chang, Hsiao-Fen G; Wang, Hwa-Tang T; Balasuriya, Udeni B R

    2016-08-01

    Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of horses. Most importantly, EAV induces abortion in pregnant mares and can establish persistent infection in up to 10-70% of the infected stallions, which will continue to shed the virus in their semen. The objective of this study was to develop and evaluate a reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) for the detection of EAV in semen and tissue samples. The newly developed assay had a limit of detection of 10 RNA copies and a 10-fold higher sensitivity than a previously described real-time RT-PCR (RT-qPCR). Evaluation of 125 semen samples revealed a sensitivity and specificity of 98.46% and 100.00%, respectively for the RT-qPCR assay, and 100.00% and 98.33%, respectively for the RT-iiPCR assay. Both assays had the same accuracy (99.2%, k=0.98) compared to virus isolation. Corresponding values derived from testing various tissue samples (n=122) collected from aborted fetuses, foals, and EAV carrier stallions are as follows: relative sensitivity, specificity, and accuracy of 88.14%, 96.83%, and 92.62% (k=0.85), respectively for the RT-qPCR assay, and 98.31%, 92.06%, and 95.08% (k=0.90), respectively for the RT-iiPCR assay. These results indicate that RT-iiPCR is a sensitive, specific, and a robust test enabling detection of EAV in semen and tissue samples with very considerable accuracy. Even though the RT-qPCR assay showed a sensitivity and specificity equal to virus isolation for semen samples, its diagnostic performance was somewhat limited for tissue samples. Thus, this new RT-iiPCR could be considered as an alternative tool in the implementation of EAV control and prevention strategies.

  20. Implementation of a microwave-assisted tissue-processing system and an automated embedding system for breast needle core biopsy samples: morphology, immunohistochemistry, and FISH evaluation.

    PubMed

    Pegolo, Enrico; Pandolfi, Maura; Di Loreto, Carla

    2013-07-01

    A platform composed of a microwave (MW)-assisted tissue-processing system and an automated embedding system has been recently introduced in pathology laboratories. Needle core biopsy (NCB) is an established, highly accurate method for diagnosing breast lesions and for providing important pathologic, predictive, and prognostic information such as biomarker expression in case of breast carcinoma. The aim of this study was to evaluate whether breast NCBs processed with the MW-assisted tissue-processing system and automatically embedded show good-quality histology preparations and whether they are suitable for the assessment of estrogen receptor (ER), progesterone receptor (PR), Ki-67, and HER2 in breast carcinoma. A series of 233 consecutive breast NCBs processed by both conventional and MW-assisted tissue-processing systems was included in this study. The histomorphologic and immunohistochemical quality, as well as the results of the evaluation of the biomarkers, were compared-the conventional processing method being the gold standard for comparison. The quality of hematoxylin-eosin and immunohistochemical tissue sections provided by the new system is comparable to that obtained after the conventional processing method. Moreover, in breast carcinomas, a perfect agreement between the paired tissues when evaluating ER and PR status (Cohen κ = 1) and a very good agreement when evaluating Ki-67 (κ = 0.91) and HER2 (κ = 0.93) have been found. In conclusion, applying strict criteria in tissue-handling steps, breast NCB can be processed and automatically embedded with these platforms. The diagnosability and the evaluation of the main prognostic and predictive biomarkers have been proved to be reliable.

  1. In-vitro micro-Raman study of tissue samples for detecting cervical and ovarian cancer with 785-nm laser excitation

    NASA Astrophysics Data System (ADS)

    Sharma, S. K.; Kamemoto, L. E.; Misra, A. K.; Goodman, M. T.; Luk, H. W.; Killeen, J. L.

    2010-04-01

    We present results of in vitro micro-Raman spectroscopy of normal and cancerous cervical and ovarian tissues excited with 785 nm near-infrared (NIR) laser. Micro- Raman spectra of squamous cervical cells of both cervix and ovarian tissues show significant differences in the spectra of normal and cancerous cells. In particular, several well-defined Raman peaks in the 775-975 cm-1 region are observed in the spectra of normal cervix squamous cells but are completely missing in the spectra of invasive cervical cancer cells. In the high-frequency 2800-3100 cm-1 region it is shown that the peak area under CH stretching band is much lower than the corresponding area in the spectra of normal cells. In the case of ovarian tissues, the micro-Raman spectra show noticeable spectral differences between normal cells and ovarian serous cancer cells. In particular, we observed the accumulation of β-carotene in ovarian serous cancer cells compared to normal ovarian cells from women with no ovarian cancer. The NIR micro-Raman spectroscopy offers a potential molecular technique for detecting cervical and ovarian cancer from the respective tissues.

  2. Quantitation of repaglinide and metabolites in mouse whole-body thin tissue sections using droplet-based liquid microjunction surface sampling-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

    DOE PAGES

    Chen, Weiqi; Wang, Lifei; Van Berkel, Gary J.; ...

    2015-11-03

    Herein, quantitation aspects of a fully automated autosampler/HPLC-MS/MS system applied for unattended droplet-based surface sampling of repaglinide dosed thin tissue sections with subsequent HPLC separation and mass spectrometric analysis of parent drug and various drug metabolites was studied. Major organs (brain, lung, liver, kidney, muscle) from whole-body thin tissue sections and corresponding organ homogenates prepared from repaglinide dosed mice were sampled by surface sampling and by bulk extraction, respectively, and analyzed by HPLC-MS/MS. A semi-quantitative agreement between data obtained by surface sampling and that by employing organ homogenate extraction was observed. Drug concentrations obtained by the two methods followed themore » same patterns for post-dose time points (0.25, 0.5, 1 and 2 h). Drug amounts determined in the specific tissues was typically higher when analyzing extracts from the organ homogenates. Furthermore, relative comparison of the levels of individual metabolites between the two analytical methods also revealed good semi-quantitative agreement.« less

  3. Quantitation of repaglinide and metabolites in mouse whole-body thin tissue sections using droplet-based liquid microjunction surface sampling-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

    SciTech Connect

    Chen, Weiqi; Wang, Lifei; Van Berkel, Gary J.; Gan, Jinping; Kertesz, Vilmos

    2015-11-03

    Herein, quantitation aspects of a fully automated autosampler/HPLC-MS/MS system applied for unattended droplet-based surface sampling of repaglinide dosed thin tissue sections with subsequent HPLC separation and mass spectrometric analysis of parent drug and various drug metabolites was studied. Major organs (brain, lung, liver, kidney, muscle) from whole-body thin tissue sections and corresponding organ homogenates prepared from repaglinide dosed mice were sampled by surface sampling and by bulk extraction, respectively, and analyzed by HPLC-MS/MS. A semi-quantitative agreement between data obtained by surface sampling and that by employing organ homogenate extraction was observed. Drug concentrations obtained by the two methods followed the same patterns for post-dose time points (0.25, 0.5, 1 and 2 h). Drug amounts determined in the specific tissues was typically higher when analyzing extracts from the organ homogenates. Furthermore, relative comparison of the levels of individual metabolites between the two analytical methods also revealed good semi-quantitative agreement.

  4. Cyanovanadate(III) complexes as novel additives for efficient generation of volatile cadmium species in complex samples prior to determinations by inductively coupled plasma mass spectrometry (ICP-MS).

    PubMed

    Yilmaz, Vedat; Arslan, Zikri; Rose, LaKeysha; Little, Maria D

    2013-10-15

    A new method has been described for generation of volatile species of Cd using vanadium(III) cyanide complex. Aqueous solutions of 0.04 mol L(-1) vanadium chloride (VCl3) and 0.12 mol L(-1) potassium cyanide (KCN) were reacted on-line yielding a suspension of vanadium hydroxide, V(OH)3. This suspension was dissolved along the stream of sample solution in dilute HCl to form heptacyanovanadate(III) complex, [V(CN)7]4-. Volatile Cd species were generated by reacting the stream of sample solution and cyanovanadate(III) complex with sodium borohydride (NaBH4). Feasibility of off-line and on-online approaches was investigated for quantitative determinations. Better precision and daily stability were achieved with on-line settings. Optimum signals were obtained from sample solutions within a range of 3 to 5% v/v HCl. A concentration of 2% m/v NaBH4 was adequate to achieve an enhancement of 20-fold in the presence of cyanovanadate(III) complex. The limits of detection were 5.0 and 4.5 ng L(-1) for 110Cd and 111Cd isotopes, respectively. Precision (%RSD) was better than 4.7% for six replicate measurements. The interferences of Cu(II) and Ni(II) were marginal (<10%) at 1.0 µg mL(-1). Depressive effects from Bi, Se and Sn were not significant below 0.1 µg mL(-1). The method was validated by determination of Cd using ICP-MS in certified reference materials of Nearshore seawater (CASS-4), Bone ash (SRM 1400), Dogfish liver (DOLT-4) and Mussel tissue (SRM 2976).

  5. Radiometric assay for phenylethanolamine N-methyltransferase and catechol O-methyltransferase in a single tissue sample: application to rat hypothalamic nuclei, pineal gland, and heart

    SciTech Connect

    Culman, J.; Torda, T.; Weise, V.K.

    1987-08-01

    A simple and highly sensitive method for simultaneous assay of phenylethanolamine N-methyltransferase (PNMT) and catechol O-methyltransferase (COMT) is described. These enzymes are determined in a single tissue homogenate using S-(methyl-/sup 3/H) adenosyl-L-methionine as methyl donor and sequentially incubating with the substrates phenylethanolamine and epinephrine. The radioactive products of the enzymatic reactions, N-methylphenylethanolamine and metanephrine, are extracted and then separated by thin-layer chromatography. The identity of the reaction products has been established chromatographically and the conditions for both enzymatic reactions in the assay procedure have been defined. Measurement of PNMT activity in the rat pineal gland or in minute fragments of other tissues (e.g., brain nuclei) has not been possible using previously described methods. Activities of PNMT and COMT in the rat pineal gland, various hypothalamic nuclei, and the auricular and ventricular myocardia are herein reported.

  6. Potlining Additives

    SciTech Connect

    Rudolf Keller

    2004-08-10

    In this project, a concept to improve the performance of aluminum production cells by introducing potlining additives was examined and tested. Boron oxide was added to cathode blocks, and titanium was dissolved in the metal pool; this resulted in the formation of titanium diboride and caused the molten aluminum to wet the carbonaceous cathode surface. Such wetting reportedly leads to operational improvements and extended cell life. In addition, boron oxide suppresses cyanide formation. This final report presents and discusses the results of this project. Substantial economic benefits for the practical implementation of the technology are projected, especially for modern cells with graphitized blocks. For example, with an energy savings of about 5% and an increase in pot life from 1500 to 2500 days, a cost savings of $ 0.023 per pound of aluminum produced is projected for a 200 kA pot.

  7. Phosphazene additives

    DOEpatents

    Harrup, Mason K; Rollins, Harry W

    2013-11-26

    An additive comprising a phosphazene compound that has at least two reactive functional groups and at least one capping functional group bonded to phosphorus atoms of the phosphazene compound. One of the at least two reactive functional groups is configured to react with cellulose and the other of the at least two reactive functional groups is configured to react with a resin, such as an amine resin of a polycarboxylic acid resin. The at least one capping functional group is selected from the group consisting of a short chain ether group, an alkoxy group, or an aryloxy group. Also disclosed are an additive-resin admixture, a method of treating a wood product, and a wood product.

  8. Assessment of ultrasound-assisted extraction as sample pre-treatment for the measurement of lead isotope ratios in marine biological tissues by multicollector inductively coupled plasma-mass spectrometry

    NASA Astrophysics Data System (ADS)

    Costas-Rodríguez, M.; Lavilla, Isela; Bendicho, Carlos

    2011-06-01

    In this work, ultrasound-assisted extraction (UAE) was evaluated as a sample preparation procedure for lead isotope ratio measurements in marine biological tissues by multicollector inductively coupled plasma-mass spectrometry. 20 mg of marine biological tissue and 1 mL of acid extractant were sonicated for 3 min at 60% ultrasound amplitude. Matrix separation was performed in the supernatant using a chromatographic exchange resin (Sr-Spec™). Total elimination of organic matter was achieved during the separation step. Microwave-assisted digestion and dry-ashing were used for comparative purposes. No significant differences were found in lead isotope ratios at 95% of confidence level. UAE emerges as an advantageous alternative to classical methods for sample preparation owing to its simplicity and rapidity ( i.e. operation steps were reduced), low reagent consumption and low contamination risks.

  9. Surfactant-aided precipitation/on-pellet-digestion (SOD) procedure provides robust and rapid sample preparation for reproducible, accurate and sensitive LC/MS quantification of therapeutic protein in plasma and tissues.

    PubMed

    An, Bo; Zhang, Ming; Johnson, Robert W; Qu, Jun

    2015-04-07

    For targeted protein quantification by liquid chromatography mass spectrometry (LC/MS), an optimal approach for efficient, robust and hi-throughput sample preparation is critical, but often remains elusive. Here we describe a straightforward surfactant-aided-precipitation/on-pellet-digestion (SOD) strategy that provides effective sample cleanup and enables high and constant peptide yields in various matrices, allowing reproducible, accurate and sensitive protein quantification. This strategy was developed using quantification of monocolnocal antibody in tissues and plasma as the model system. Surfactant treatment before precipitation substantially increased peptide recovery and reproducibility from plasma/tissue, likely because surfactant permits extensive denaturation/reduction/alkylation of proteins and inactivation of endogenous protease inhibitors, and facilitates removal of matrix components. The subsequent precipitation procedure effectively eliminates the surfactant and nonprotein matrix components, and the thorough denaturation by both surfactant and precipitation enabled very rapid on-pellet-digestion (45 min at 37 °C) with high peptide recovery. The performance of SOD was systematically compared against in-solution-digestion, in-gel-digestion and filter-aided-sample-preparation (FASP) in plasma/tissues, and then examined in a full pharmacokinetic study in rats. SOD achieved the best peptide recovery (∼21.0-700% higher than the other three methods across various matrices), reproducibility (3.75-10.9%) and sensitivity (28-30 ng/g across plasma and tissue matrices), and its performance was independent of matrix types. Finally, in validation and pharmacokinetic studies in rats, SOD outperformed other methods and provided highly accurate and precise quantification in all plasma samples without using stable isotope labeled (SIL)-protein internal standard (I.S.). In summary, the SOD method has proven to be highly robust, efficient and rapid, making it readily

  10. A comparison of 3D poly(ε-caprolactone) tissue engineering scaffolds produced with conventional and additive manufacturing techniques by means of quantitative analysis of SR μ-CT images

    NASA Astrophysics Data System (ADS)

    Brun, F.; Intranuovo, F.; Mohammadi, S.; Domingos, M.; Favia, P.; Tromba, G.

    2013-07-01

    The technique used to produce a 3D tissue engineering (TE) scaffold is of fundamental importance in order to guarantee its proper morphological characteristics. An accurate assessment of the resulting structural properties is therefore crucial in order to evaluate the effectiveness of the produced scaffold. Synchrotron radiation (SR) computed microtomography (μ-CT) combined with further image analysis seems to be one of the most effective techniques to this aim. However, a quantitative assessment of the morphological parameters directly from the reconstructed images is a non trivial task. This study considers two different poly(ε-caprolactone) (PCL) scaffolds fabricated with a conventional technique (Solvent Casting Particulate Leaching, SCPL) and an additive manufacturing (AM) technique (BioCell Printing), respectively. With the first technique it is possible to produce scaffolds with random, non-regular, rounded pore geometry. The AM technique instead is able to produce scaffolds with square-shaped interconnected pores of regular dimension. Therefore, the final morphology of the AM scaffolds can be predicted and the resulting model can be used for the validation of the applied imaging and image analysis protocols. It is here reported a SR μ-CT image analysis approach that is able to effectively and accurately reveal the differences in the pore- and throat-size distributions as well as connectivity of both AM and SCPL scaffolds.

  11. Rapid extraction and reverse phase-liquid chromatographic separation of mercury(II) and methylmercury in fish samples with inductively coupled plasma mass spectrometric detection applying oxygen addition into plasma.

    PubMed

    Döker, Serhat; Boşgelmez, İffet İpek

    2015-10-01

    A simple and sensitive procedure was developed for extraction and speciation of mercury in fish. Species separation was accomplished with reverse phase-high performance liquid chromatography (HPLC) hyphenated to inductively coupled plasma mass spectrometry (ICP-MS). Oxygen addition into plasma allowed use of organic-rich mobile phase, achieving species separation in 4 min. Mercury species extraction was achieved by microwave exposure for 2 min at mild conditions (60°C, pH 2.0), avoiding necessity of neutralizing sample prior to injection in HPLC, and reducing number of sample preparation steps, analytical source of errors and inter conversion of species. Limit of detection for entire procedure was found to be 0.2 and 0.1 ng g(-1) for mercuric ion and methylmercury, respectively. The method was applied to certified reference materials (TORT-2 and DORM-2) and commercialized fish samples (Mullus barbatus, Sparus aurata, Trachurus mediterraneus, Mugil soiuy, Dicentrarchus labrax, and Pomatomus saltatrix) from Black Sea.

  12. Evaluation of a real-time PCR and a loop-mediated isothermal amplification for detection of Xanthomonas arboricola pv. pruni in plant tissue samples.

    PubMed

    Palacio-Bielsa, Ana; López-Soriano, Pablo; Bühlmann, Andreas; van Doorn, Joop; Pham, Khanh; Cambra, Miguel A; Berruete, Isabel M; Pothier, Joël F; Duffy, Brion; Olmos, Antonio; López, María M

    2015-05-01

    Operational capacity of real-time PCR and loop-mediated isothermal amplification (LAMP) diagnostic assays for detection of Xanthomonas arboricola pv. pruni was established in a ring-test involving four laboratories. Symptomatic and healthy almond leaf samples with two methods of sample preparation were analyzed. Kappa coefficient, sensitivity, specificity, likelihood ratios and post-test probability of detection were estimated to manage the risk associated with the use of the two methods.

  13. A procedure for tissue freezing and processing applicable to both intra-operative frozen section diagnosis and tissue banking in surgical pathology.

    PubMed

    Steu, Susanne; Baucamp, Maya; von Dach, Gabriela; Bawohl, Marion; Dettwiler, Susanne; Storz, Martina; Moch, Holger; Schraml, Peter

    2008-03-01

    Different methods for snap freezing surgical human tissue specimens exist. At pathology institutes with higher work loads, solid carbon dioxide, freezing sprays, and cryostat freezing are commonly used as coolants for diagnosing frozen tissue sections, whereas for tissue banking, liquid nitrogen or isopentane cooled with liquid nitrogen is preferred. Freezing tissues for diagnostic and research purposes are therefore often time consuming, laborious, even hazardous, and not user friendly. In tissue banks, frozen tissue samples are stored in cryovials, capsules, cryomolds, or cryocassettes. Tissues are additionally embedded using freezing media or wrapped in plastic bags or aluminum foils to prevent desiccation. The latter method aggravates enormously further tissue handling and processing. Here, we describe an isopentane-based workflow which concurrently facilitates tissue freezing and processing for both routine intra-operative frozen section and tissue banking and satisfies the qualitative demands of pathologists, cancer researchers, laboratory technicians, and tissue bankers.

  14. Effects of Supplemental Acerola Juice on the Mineral Concentrations in Liver and Kidney Tissue Samples of Mice Fed with Cafeteria Diet.

    PubMed

    Leffa, Daniela Dimer; dos Santos, Carla Eliete Iochims; Daumann, Francine; Longaretti, Luiza Martins; Amaral, Livio; Dias, Johnny Ferraz; da Silva, Juliana; Andrade, Vanessa Moraes

    2015-09-01

    We evaluated the impact of a supplemental acerola juice (unripe, ripe, and industrial) and its main pharmaceutically active components on the concentrations of minerals in the liver and kidney of mice fed with cafeteria diet. Swiss male mice were fed with a cafeteria (CAF) diet for 13 weeks. The CAF consisted of a variety of supermarket products with high energy content. Subsequently, animals received one of the following food supplements for 1 month: water, unripe acerola juice, ripe acerola juice, industrial acerola juice, vitamin C, or rutin. Mineral concentrations of the tissues were determined by particle-induced X-ray emission (PIXE). Our study suggests that the simultaneous intake of acerola juices, vitamin C, or rutin in association with a hypercaloric and hyperlipidic diet provides change in the mineral composition of organisms in the conditions of this study, which plays an important role in the antioxidant defenses of the body. This may help to reduce the metabolism of the fat tissue or even to reduce the oxidative stress.

  15. A modified ‘NanoSuit®’ preserves wet samples in high vacuum: direct observations on cells and tissues in field-emission scanning electron microscopy

    PubMed Central

    Takaku, Yasuharu; Suzuki, Hiroshi; Kawasaki, Hideya; Ohta, Isao; Ishii, Daisuke; Hirakawa, Satoshi; Tsutsui, Takami; Matsumoto, Haruko; Takehara, Sayuri; Nakane, Chinatsu; Sakaida, Kana; Suzuki, Chiaki; Muranaka, Yoshinori; Kikuchi, Hirotoshi; Konno, Hiroyuki; Shimomura, Masatsugu

    2017-01-01

    Although field-emission scanning electron microscopy (FE-SEM) has proven very useful in biomedical research, the high vacuum required (10−3 to 10−7 Pa) precludes direct observations of living cells and tissues at high resolution and often produces unwanted structural changes. We have previously described a method that allows the investigator to keep a variety of insect larvae alive in the high vacuum environment of the electron microscope by encasing the organisms in a thin, vacuum-proof suit, the ‘NanoSuit®'. However, it was impossible to protect wet tissues freshly excised from intact organisms or cultured cells. Here we describe an improved ‘NanoSuit' technique to overcome this limitation. We protected the specimens with a surface shield enhancer (SSE) solution that consists of glycerine and electrolytes and found that the fine structure of the SSE-treated specimens is superior to that of conventionally prepared specimens. The SSE-based NanoSuit affords a much stronger barrier to gas and/or liquid loss than the previous NanoSuit did and, since it allows more detailed images, it could significantly help to elucidate the ‘real' organization of cells and their functions.

  16. [Determination of cobalt in small samples of tissues and organs of the rainbow trout (Salmo gairdneri) by flameless atomic-absorption-spectroscopy (author's transl)].

    PubMed

    Harms, U; Kunze, J

    1977-07-29

    In order to increase our knowledge about the distribution pattern of the trace mineral cobalt in fish, cobalt contents of tissues and inner organs of rainbow trout were analysed. Cobalt determinations were performed by flameless atomic-absorption-spectroscopy with a heated graphite analyzer after digestion of the organic material with concentrated nitric acid in a closed system under pressure (pressure decomposition). In order to concentrate and separate the trace cobalt quantities from the main matrix constituents a micro-solvent-extraction system with ammonium pyrrolidinedithiocarbamate (APDC) as heavy metal chelating reagent and toluene as extraction solvent was developed. The extraction procedure yielded a recovery of more than 95% as determined the use of an isotope method (radiotracer experiments with Co-57). Results (calculated on wet weight basis) showed that the element under study was mainly accumulated in the kidney (0.195-0.449 microgram/g). Smaller amounts were found in blood (0.038-0.090 microgram/g), spleen (0.015-0.078 microgram/g, and liver 0.015-0.068 microgram/g). The values for skeletal and muscle tissue were low and ranged from 0.007 to 0.014 microgram/g a 0.002 to 0.007 microgram/g respectively.

  17. Micropatterned, clickable culture substrates enable in situ spatiotemporal control of human PSC-derived neural tissue morphology† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4cc08665a Click here for additional data file.

    PubMed Central

    Knight, G. T.; Sha, J.

    2015-01-01

    We describe a modular culture platform that enables spatiotemporal control of the morphology of 2D neural tissues derived from human pluripotent stem cells (hPSCs) by simply adding clickable peptides to the media. It should be widely applicable for elucidating how spatiotemporal changes in morphology and substrate biochemistry regulate tissue morphogenesis. PMID:25688384

  18. Risk-based approach to developing a national residue sampling plan for testing under European Union regulation for veterinary medicinal products and coccidiostat feed additives in domestic animal production.

    PubMed

    Danaher, Martin; Shanahan, Conor; Butler, Francis; Evans, Rhodri; O'Sullivan, Dan; Glynn, Denise; Camon, Tim; Lawlor, Peadar; O'Keeffe, Michael

    2016-07-01

    A ranking system for veterinary medicinal products and coccidiostat feed additives has been developed as a tool to be applied in a risk-based approach to the residue testing programme for foods of animal origin in the Irish National Residue Control Plan (NRCP). Three characteristics of substances that may occur as residues in food are included in the developed risk ranking system: Potency, as measured by the acceptable daily intake assigned by the European Medicines Agency Committee for Medicinal Products for Veterinary Use, to each substance; Usage, as measured by the three factors of Number of Doses, use on Individual animals or for Group treatment, and Withdrawal Period; and Residue Occurrence, as measured by the number of Non-Compliant Samples in the NRCP. For both Number of Doses and Non-Compliant Samples, data for the 5-year period 2008-12 have been used. The risk ranking system for substances was developed for beef cattle, sheep and goats, pigs, chickens and dairy cattle using a scoring system applied to the various parameters described above to give an overall score based on the following equation: Potency × Usage (Number of Doses + Individual/Group Use + Withdrawal Period) × Residue Occurrence. Applying this risk ranking system, the following substances are ranked very highly: antimicrobials such as amoxicillin (for all species except pigs), marbofloxacillin (for beef cattle), oxytetracycline (for all species except chickens), sulfadiazine with trimethoprim (for pigs and chickens) and tilmicosin (for chickens); antiparasitic drugs, such as the benzimidazoles triclabendazole (for beef and dairy cattle), fenbendazole/oxfendazole (for sheep/goats and dairy cattle) and albendazole (for dairy cattle), the avermectin ivermectin (for beef cattle), and anti-fluke drugs closantel and rafoxanide (for sheep/goats); the anticoccidials monensin, narasin, nicarbazin and toltrazuril (for chickens). The risk ranking system described is a relatively simple system

  19. Simultaneous determination of nickel and copper by H-point standard addition method-first-order derivative spectrophotometry in plant samples after separation and preconcentration on modified natural clinoptilolite as a new sorbent.

    PubMed

    Roohparvar, Rasool; Taher, Mohammad Ali; Mohadesi, Alireza

    2008-01-01

    For the simultaneous determination of nickel(ll) and copper(ll) in plant samples, a rapid and accurate method was developed. In this method, solid-phase extraction (SPE) and first-order derivative spectrophotometry (FDS) are combined, and the result is coupled with the H-point standard addition method (HPSAM). Compared with normal spectrophotometry, derivative spectrophotometry offers the advantages of increased selectivity and sensitivity. As there is no need for carrying out any pretreatment of the sample, the spectrophotometry method is easy, but because of a high detection limit, it is not so practical. In order to decrease the detection limit, it is suggested to combine spectrophotometry with a preconcentration method such as SPE. In the present work, after separation and preconcentration of Ni(ll) and Cu(ll) on modified clinoptilolite zeolite that is loaded with 2-[1-(2-hydroxy-5-sulforphenyl)-3-phenyl-5-formaza-no]-benzoic acid monosodium salt (zincon) as a selective chromogenic reagent, FDS-HPSAM, which is a simple and selective spectrophotometric method, has been applied for simultaneous determination of these ions. With optimum conditions, the detection limit in original solutions is 0.7 and 0.5 ng/mL, respectively, for nickel and copper. The linear concentration ranges in the proposed method for nickel and copper ions in original solutions are 1.1 to 3.0 x 10(3) and 0.9 to 2.0 x 10(3) ng/mL, respectively. The recommended procedure is applied to successful determination of Cu(ll) and Ni(ll) in standard and real samples.

  20. Carotid Catheterization and Automated Blood Sampling Induce Systemic IL-6 Secretion and Local Tissue Damage and Inflammation in the Heart, Kidneys, Liver and Salivary Glands in NMRI Mice

    PubMed Central

    Teilmann, Anne Charlotte; Rozell, Björn; Kalliokoski, Otto; Hau, Jann; Abelson, Klas S. P.

    2016-01-01

    Automated blood sampling through a vascular catheter is a frequently utilized technique in laboratory mice. The potential immunological and physiological implications associated with this technique have, however, not been investigated in detail. The present study compared plasma levels of the cytokines IL-1β, IL-2, IL-6, IL-10, IL-17A, GM-CSF, IFN-γ and TNF-α in male NMRI mice that had been subjected to carotid artery catheterization and subsequent automated blood sampling with age-matched control mice. Body weight and histopathological changes in the surgical area, including the salivary glands, the heart, brain, spleen, liver, kidneys and lungs were compared. Catheterized mice had higher levels of IL-6 than did control mice, but other cytokine levels did not differ between the groups. No significant difference in body weight was found. The histology revealed inflammatory and regenerative (healing) changes at surgical sites of all catheterized mice, with mild inflammatory changes extending into the salivary glands. Several catheterized mice had multifocal degenerative to necrotic changes with inflammation in the heart, kidneys and livers, suggesting that thrombi had detached from the catheter tip and embolized to distant sites. Thus, catheterization and subsequent automated blood sampling may have physiological impact. Possible confounding effects of visceral damage should be assessed and considered, when using catheterized mouse models. PMID:27832170

  1. Polylactides in additive biomanufacturing.

    PubMed

    Poh, Patrina S P; Chhaya, Mohit P; Wunner, Felix M; De-Juan-Pardo, Elena M; Schilling, Arndt F; Schantz, Jan-Thorsten; van Griensven, Martijn; Hutmacher, Dietmar W

    2016-12-15

    New advanced manufacturing technologies under the alias of additive biomanufacturing allow the design and fabrication of a range of products from pre-operative models, cutting guides and medical devices to scaffolds. The process of printing in 3 dimensions of cells, extracellular matrix (ECM) and biomaterials (bioinks, powders, etc.) to generate in vitro and/or in vivo tissue analogue structures has been termed bioprinting. To further advance in additive biomanufacturing, there are many aspects that we can learn from the wider additive manufacturing (AM) industry, which have progressed tremendously since its introduction into the manufacturing sector. First, this review gives an overview of additive manufacturing and both industry and academia efforts in addressing specific challenges in the AM technologies to drive toward AM-enabled industrial revolution. After which, considerations of poly(lactides) as a biomaterial in additive biomanufacturing are discussed. Challenges in wider additive biomanufacturing field are discussed in terms of (a) biomaterials; (b) computer-aided design, engineering and manufacturing; (c) AM and additive biomanufacturing printers hardware; and (d) system integration. Finally, the outlook for additive biomanufacturing was discussed.

  2. Is there a role for mammary stem cells in inflammatory breast carcinoma?: a review of evidence from cell line, animal model, and human tissue sample experiments.

    PubMed

    Van Laere, Steven; Limame, Ridha; Van Marck, Eric A; Vermeulen, Peter B; Dirix, Luc Y

    2010-06-01

    Stem cells are pluripotent cells, with a large replicative potential, which perform normal physiological functions such as tissue renewal and damage repair. However, because of their long lifespan and high replicative potential, stem cells are ideal targets to accumulate multiple mutations. Therefore, they can be regarded as being responsible for the initiation of tumor formation. In the past, numerous studies have shown that the presence of an elaborate stem cell compartment within a tumor is associated with aggressive tumor cell behavior, frequent formation of metastases, resistance to therapy, and poor patient survival. From this perspective, tumors from patients with inflammatory breast cancer (IBC), an aggressive breast cancer subtype with a dismal clinical course, are most likely to be associated with stem cell biology. To date, this hypothesis is corroborated by evidence resulting from in vitro and in vivo experiments. Both gene and microRNA expression profiles highlighted several stem cell-specific signal transduction pathways that are hyperactivated in IBC. Also, these stem cell-specific signal transduction pathways seem to converge in the activation of nuclear factor-kappa B, a molecular hallmark of IBC, and induction of epithelial-to-mesenchymal transition. Recently, the latter mechanism was identified as a prerequisite for the induction of stem cell characteristics in breast cancer cells.

  3. Effect of Layer Thickness and Printing Orientation on Mechanical Properties and Dimensional Accuracy of 3D Printed Porous Samples for Bone Tissue Engineering

    PubMed Central

    Farzadi, Arghavan; Solati-Hashjin, Mehran; Asadi-Eydivand, Mitra; Abu Osman, Noor Azuan

    2014-01-01

    Powder-based inkjet 3D printing method is one of the most attractive solid free form techniques. It involves a sequential layering process through which 3D porous scaffolds can be directly produced from computer-generated models. 3D printed products' quality are controlled by the optimal build parameters. In this study, Calcium Sulfate based powders were used for porous scaffolds fabrication. The printed scaffolds of 0.8 mm pore size, with different layer thickness and printing orientation, were subjected to the depowdering step. The effects of four layer thicknesses and printing orientations, (parallel to X, Y and Z), on the physical and mechanical properties of printed scaffolds were investigated. It was observed that the compressive strength, toughness and Young's modulus of samples with 0.1125 and 0.125 mm layer thickness were more than others. Furthermore, the results of SEM and μCT analyses showed that samples with 0.1125 mm layer thickness printed in X direction have more dimensional accuracy and significantly close to CAD software based designs with predefined pore size, porosity and pore interconnectivity. PMID:25233468

  4. Effects of TGF-beta signalling inhibition with galunisertib (LY2157299) in hepatocellular carcinoma models and in ex vivo whole tumor tissue samples from patients

    PubMed Central

    Serova, Maria; Tijeras-Raballand, Annemilaï; Santos, Célia Dos; Albuquerque, Miguel; Paradis, Valerie; Neuzillet, Cindy; Benhadji, Karim A.; Raymond, Eric; Faivre, Sandrine; de Gramont, Armand

    2015-01-01

    Galunisertib (LY2157299) is a selective ATP-mimetic inhibitor of TGF-β receptor (TβR)-I activation currently under clinical investigation in hepatocellular carcinoma (HCC) patients. Our study explored the effects of galunisertib in vitro in HCC cell lines and ex vivo on patient samples. Galunisertib was evaluated in HepG2, Hep3B, Huh7, JHH6 and SK-HEP1 cells as well as in SK-HEP1-derived cells tolerant to sorafenib (SK-Sora) and sunitinib (SK-Suni). Exogenous stimulation of all HCC cell lines with TGF-β yielded downstream activation of p-Smad2 and p-Smad3 that was potently inhibited with galunisertib treatment at micromolar concentrations. Despite limited antiproliferative effects, galunisertib yielded potent anti-invasive properties. Tumor slices from 13 patients with HCC surgically resected were exposed ex vivo to 1 μM and 10 μM galunisertib, 5 μM sorafenib or a combination of both drugs for 48 hours. Galunisertib but not sorafenib decreased p-Smad2/3 downstream TGF-β signaling. Immunohistochemistry analysis of galunisertib and sorafenib-exposed samples showed a significant decrease of the proliferative marker Ki67 and increase of the apoptotic marker caspase-3. In combination, galunisertib potentiated the effect of sorafenib efficiently by inhibiting proliferation and increasing apoptosis. Our data suggest that galunisertib may be active in patients with HCC and could potentiate the effects of sorafenib. PMID:26057634

  5. Effect of layer thickness and printing orientation on mechanical properties and dimensional accuracy of 3D printed porous samples for bone tissue engineering.

    PubMed

    Farzadi, Arghavan; Solati-Hashjin, Mehran; Asadi-Eydivand, Mitra; Abu Osman, Noor Azuan

    2014-01-01

    Powder-based inkjet 3D printing method is one of the most attractive solid free form techniques. It involves a sequential layering process through which 3D porous scaffolds can be directly produced from computer-generated models. 3D printed products' quality are controlled by the optimal build parameters. In this study, Calcium Sulfate based powders were used for porous scaffolds fabrication. The printed scaffolds of 0.8 mm pore size, with different layer thickness and printing orientation, were subjected to the depowdering step. The effects of four layer thicknesses and printing orientations, (parallel to X, Y and Z), on the physical and mechanical properties of printed scaffolds were investigated. It was observed that the compressive strength, toughness and Young's modulus of samples with 0.1125 and 0.125 mm layer thickness were more than others. Furthermore, the results of SEM and μCT analyses showed that samples with 0.1125 mm layer thickness printed in X direction have more dimensional accuracy and significantly close to CAD software based designs with predefined pore size, porosity and pore interconnectivity.

  6. Assessing the function of mitochondria in cytosolic context in human skeletal muscle: adopting high-resolution respirometry to homogenate of needle biopsy tissue samples.

    PubMed

    Ziak, Jakub; Krajcova, Adela; Jiroutkova, Katerina; Nemcova, Vlasta; Dzupa, Valer; Duska, Frantisek

    2015-03-01

    Using skeletal muscle homogenates for respirometry has many advantages, but the main challenge is avoiding the damage to outer mitochondrial membrane (OMM) and complex I. By optimising the amount of muscle and careful titration of substrates and inhibitors we developed a new protocol and compared it to isolated mitochondria. We found acceptable damage to OMM (~10-15% increment of oxygen flux after addition of cytochrome c) and to complex I (~70% of electron flux). Homogenate retained ~90% of phosphorylation capacity of isolated mitochondria. The use of fresh homogenate was crucial as mitochondrial function declined rapidly after 2-3h of cold storage.

  7. Determination of selenium in biological tissue samples rich in phosphorus using electrothermal atomization with Zeeman-effect background correction and (NH 4) 3RhCl 6+citric acid as a mixed chemical modifier

    NASA Astrophysics Data System (ADS)

    Mei, Li; Zhe-ming, Ni; Zhu, Rao

    1998-09-01

    Spectral interferences from phosphorus on the determination of selenium in biological tissue materials were not observed when a Zeeman-effect background correction was used with rhodium as a chemical modifier. A suppression effect on the selenium signal resulted when the concentration of phosphorus present was greater than 1.0 mg ml -1. Rhodium was found to be more effective than palladium in overcoming the phosphate interference. Analytical procedures for the direct determination of trace selenium in standard reference materials by graphite furnace atomic absorption spectrometry following sample dissolution in nitric acid and hydrogen peroxide using a microwave oven has been described. The results obtained agreed favourably with the certified values.

  8. Histopathological findings, phenotyping of inflammatory cells, and expression of markers of nitritative injury in joint tissue samples from calves after vaccination and intraarticular challenge with Mycoplasma bovis strain 1067

    PubMed Central

    2014-01-01

    Background The pathogenesis of caseonecrotic lesions developing in lungs and joints of calves infected with Mycoplasma bovis is not clear and attempts to prevent M. bovis-induced disease by vaccines have been largely unsuccessful. In this investigation, joint samples from 4 calves, i.e. 2 vaccinated and 2 non-vaccinated, of a vaccination experiment with intraarticular challenge were examined. The aim was to characterize the histopathological findings, the phenotypes of inflammatory cells, the expression of class II major histocompatibility complex (MHC class II) molecules, and the expression of markers for nitritative stress, i.e. inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT), in synovial membrane samples from these calves. Furthermore, the samples were examined for M. bovis antigens including variable surface protein (Vsp) antigens and M. bovis organisms by cultivation techniques. Results The inoculated joints of all 4 calves had caseonecrotic and inflammatory lesions. Necrotic foci were demarcated by phagocytic cells, i.e. macrophages and neutrophilic granulocytes, and by T and B lymphocytes. The presence of M. bovis antigens in necrotic tissue lesions was associated with expression of iNOS and NT by macrophages. Only single macrophages demarcating the necrotic foci were positive for MHC class II. Microbiological results revealed that M. bovis had spread to approximately 27% of the non-inoculated joints. Differences in extent or severity between the lesions in samples from vaccinated and non-vaccinated animals were not seen. Conclusions The results suggest that nitritative injury, as in pneumonic lung tissue of M. bovis-infected calves, is involved in the development of caseonecrotic joint lesions. Only single macrophages were positive for MHC class II indicating down-regulation of antigen-presenting mechanisms possibly caused by local production of iNOS and NO by infiltrating macrophages. PMID:25162202

  9. Immunogenic HLA-DR-Presented Self-Peptides Identified Directly from Clinical Samples of Synovial Tissue, Synovial Fluid, or Peripheral Blood in Patients with Rheumatoid Arthritis or Lyme Arthritis.

    PubMed

    Wang, Qi; Drouin, Elise E; Yao, Chunxiang; Zhang, Jiyang; Huang, Yu; Leon, Deborah R; Steere, Allen C; Costello, Catherine E

    2017-01-06

    Human leukocyte antigen-antigen D related (HLA-DR) molecules are highly expressed in synovial tissue (ST), the target of the immune response in chronic inflammatory forms of arthritis. Here, we used LC-MS/MS to identify HLA-DR-presented self-peptides in cells taken directly from clinical samples: ST, synovial fluid mononuclear cells (SFMC), or peripheral blood mononuclear cells (PBMC) from five patients with rheumatoid arthritis (RA) and eight with Lyme arthritis (LA). We identified 1593 non-redundant HLA-DR-presented peptides, derived from 870 source proteins. A total of 67% of the peptides identified in SFMC and 55% of those found in PBMC were found in ST, but analysis of SFMC/PBMC also revealed new antigen-presented peptides. Peptides were synthesized and examined for reactivity with the patients' PBMC. To date, three autoantigens in RA and four novel autoantigens in LA, presented in ST and/or PBMC, were shown to be targets of T- and B-cell responses in these diseases; ongoing analyses may add to this list. Thus, immunoprecipitation and LC-MS/MS can now identify hundreds of HLA-DR-presented self-peptides from individual patients' tissues or fluids with mixed cell populations. Importantly, identification of HLA-DR-presented peptides from SFMC or PBMC allows testing of more patients, including those early in the disease. Direct analysis of clinical samples facilitates identification of novel immunogenic T-cell epitopes.

  10. Erroneous aluminum and cobalt tissue concentrations from using formalin.

    PubMed

    Meldrum, R D

    2001-10-01

    Because of the liberation of metal particles from orthopedic implants, concerns have been voiced about their long-term safety. To document the presence and measure the amount of metal in periprosthetic tissues, many reports have analyzed tissue specimens obtained from revision surgery. Some report the inclusion of formalin in tissue processing. However, the unavailability of any medical-grade formalin solution with a known metal content raises the suspicion of sample contamination. The purpose of this study was to measure the concentration of the metals in the formalin found in common orthopedic alloys and to assess this chemical's role in removing metal ions from tissue samples. To do this, tissues with known metal concentrations of cobalt and aluminum were mixed with formalin, and the differences in the formalin and tissue concentrations of these elements were evaluated. Tissue and formalin samples were tested prior to and immediately after their mixing and then at three 10-day intervals. At the end of the study additional unmixed samples of formalin and tissue for both metals were tested to ensure there had been no contamination. Both cobalt and aluminum were found in off-the-shelf formalin, with observed decreases in tissue metal concentration and increases in formalin metal concentration.

  11. Evaluation of ultrasound-assisted extraction as sample pre-treatment for quantitative determination of rare earth elements in marine biological tissues by inductively coupled plasma-mass spectrometry.

    PubMed

    Costas, M; Lavilla, I; Gil, S; Pena, F; de la Calle, I; Cabaleiro, N; Bendicho, C

    2010-10-29

    In this work, the determination of rare earth elements (REEs), i.e. Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu in marine biological tissues by inductively coupled-mass spectrometry (ICP-MS) after a sample preparation method based on ultrasound-assisted extraction (UAE) is described. The suitability of the extracts for ICP-MS measurements was evaluated. For that, studies were focused on the following issues: (i) use of clean up of extracts with a C18 cartridge for non-polar solid phase extraction; (ii) use of different internal standards; (iii) signal drift caused by changes in the nebulization efficiency and salt deposition on the cones during the analysis. The signal drift produced by direct introduction of biological extracts in the instrument was evaluated using a calibration verification standard for bracketing (standard-sample bracketing, SSB) and cumulative sum (CUSUM) control charts. Parameters influencing extraction such as extractant composition, mass-to-volume ratio, particle size, sonication time and sonication amplitude were optimized. Diluted single acids (HNO(3) and HCl) and mixtures (HNO(3)+HCl) were evaluated for improving the extraction efficiency. Quantitative recoveries for REEs were achieved using 5 mL of 3% (v/v) HNO(3)+2% (v/v) HCl, particle size <200 μm, 3 min of sonication time and 50% of sonication amplitude. Precision, expressed as relative standard deviation from three independent extractions, ranged from 0.1 to 8%. In general, LODs were improved by a factor of 5 in comparison with those obtained after microwave-assisted digestion (MAD). The accuracy of the method was evaluated using the CRM BCR-668 (mussel tissue). Different seafood samples of common consumption were analyzed by ICP-MS after UAE and MAD.

  12. Failed detection of Bovine viral diarrhea virus 2 subgenotype a (BVDV-2a) by direct fluorescent antibody test on tissue samples due to reduced reactivity of field isolates to raw anti-BVDV antibody.

    PubMed

    Yan, Lifang; Pace, Lanny W; Baughman, Brittany; Wilson, Floyd D; Zhang, Shuping; Zhang, Michael Z

    2016-03-01

    Bovine viral diarrhea virus 1 (BVDV-1) is associated with mild or subclinical infections, whereas BVDV-2 is frequently implicated in outbreaks of severe thrombocytopenia and acute fatal disease. In the present study, the carcass of a beef breed cow and tissue samples of a beef calf were received for laboratory diagnosis. Both animals exhibited severe clinical signs compatible with thrombocytopenia or hemorrhagic syndrome. Direct fluorescent antibody test (DFAT) failed to detect BVDV antigen in the tissue specimens of both cases. However, immunohistochemistry (IHC) revealed the presence of BVDV antigen in oral and esophageal mucosa and Peyer patches of the beef breed cow. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) detected BVDV-2 in selected tissues of both animals. Subsequently, BVDV was isolated from both cases and subjected to genetic and serologic characterizations. Mutations in the 5'-untranslated genomic region (5'-UTR) primer and probe binding sites and the E2 gene were associated with reduced efficiency of an established real-time RT-PCR assay and amino acid alterations in the E2 glycoprotein, respectively. Both viral isolates were classified by real-time RT-PCR and phylogenetic analysis as BVDV-2 subgenotype a. Unlike BVDV reference strains Singer and 125c, the isolates cross-reacted with anti-BVDV-1 and anti-BVDV-2 reference sera, indicating antigenic variations in field isolates. The isolates also showed reduced reactivity to porcine anti-BVDV antiserum (the raw serum used to produce BVDV DFA conjugate). In summary, data from the present investigation indicated that genetic and antigenic variations affected the performance of detection assays, especially DFAT, highlighting the need for regular evaluation and modification of BVDV tests.

  13. Quantitative determination of selenium and mercury, and an ICP-MS semi-quantitative scan of other elements in samples of eagle tissues collected from the Pacific Northwest--Summer 2011

    USGS Publications Warehouse

    May, Thomas; Walther, Mike; Brumbaugh, William

    2013-01-01

    Eagle tissues from dead eagle carcasses were collected by U.S. Fish and Wildlife Service personnel at various locations in the Pacific Northwest as part of a study to document the occurrence of metal and metalloid contaminants. A group of 182 eagle tissue samples, consisting of liver, kidney, brain, talon, feather, femur, humerus, and stomach contents, were quantitatively analyzed for concentrations of selenium and mercury by atomic absorption techniques, and for other elements by semi-quantitative scan with an inductively coupled plasma-mass spectrometer. For the various tissue matrices analyzed by an ICP-MS semiquantitative scan, some elemental concentrations (micrograms per gram dry weight) were quite variable within a particular matrix; notable observations were as follows: lead concentrations ranged from 0.2 to 31 in femurs, 0.1 to 29 in humeri, 0.1 to 54 in talons, less than (<) 0.05 to 120 in livers, <0.05 to 34 in kidneys, and 0.05 to 8 in brains; copper concentrations ranged from 5 to 9 in feathers, 8 to 47 in livers, 7 to 43 in kidneys, and 7 to 28 in brains; cadmium concentrations ranged from 0.1 to 10 in kidneys. In stomach contents, concentrations of vanadium ranged from 0.08 to 5, chromium 2 to 34, manganese 1 to 57, copper 2 to 69, arsenic <0.05 to 6, rubidium 1 to 13, and barium <0.5 to 18. Selenium concentrations from highest to lowest based on the matrix mean were as follows: kidney, liver, feather, brain, stomach content, talon, femur, and humerus. For mercury, the highest to lowest concentrations were feather, liver, talon, brain, stomach content, femur, and humerus.

  14. Identification of cDNA encoding an additional. alpha. subunit of a human GTP-binding protein: Expression of three. alpha. sub i subtypes in human tissues and cell lines

    SciTech Connect

    Kim, S.; Ang, S.L.; Bloch, D.B.; Bloch, K.D.; Kawahara, Y.; Tolman, C.; Lee, R.; Seidman, J.G.; Neer, E.J. )

    1988-06-01

    The guanine nucleotide-binding proteins (G proteins), which mediate hormonal regulation of many membrane functions, are composed of {alpha}, {beta}, and {gamma} subunits. The authors have cloned and characterized cDNA from a human T-cell library encoding a form of {alpha}{sub i} that is different from the human {alpha}{sub i} subtypes previously reported. {alpha}{sub i} is the {alpha} subunit of a class of G proteins that inhibits adenylate cyclase and regulates other enzymes and ion channels. This cDNA encodes a polypeptide of 354 amino acids and is assigned to encode the {alpha}{sub i-3} subtype of G proteins on the basis of its similarity to other {alpha}{sub i}-like cDNAs and the presence of a predicted site for ADP ribosylation by pertussis toxin. They have determined the expression of mRNA for this and two other subtypes of human {alpha}{sub i} ({alpha}{sub i-1} and {alpha}{sub i-2}) in a variety of human fetal tissues and in human cell lines. All three {alpha}{sub i} subtypes were present in the tissues tested. However, analysis of individual cell types reveals specificity of {alpha}{sub i-1} expression. mRNA for {alpha}{i-1} is absent in T cells, B cells, and monocytes but is present in other cell lines. The finding of differential expression of {alpha}{sub i-1} genes may permit characterization of distinct physiological roles for this {alpha}{sub i} subunit. mRNA for {alpha}{sub i-2} and {alpha}{sub i-3} was found in all the primary and transformed cell lines tested. Thus, some cells contain all three {alpha}{sub i} subtypes. This observation raises the question of how cells prevent cross talk among receptors that are coupled to effectors through such similar {alpha} proteins.

  15. Histological observations in the Hawaiian reef coral, Porites compressa, affected by Porites bleaching with tissue loss.

    PubMed

    Sudek, M; Work, T M; Aeby, G S; Davy, S K

    2012-10-01

    The scleractinian finger coral Porites compressa is affected by the coral disease Porites bleaching with tissue loss (PBTL). This disease initially manifests as bleaching of the coenenchyme (tissue between polyps) while the polyps remain brown with eventual tissue loss and subsequent algal overgrowth of the bare skeleton. Histopathological investigation showed a loss of symbiont and melanin-containing granular cells which was more pronounced in the coenenchyme than the polyps. Cell counts confirmed a 65% reduction in symbiont density. Tissue loss was due to tissue fragmentation and necrosis in affected areas. In addition, a reduction in putative bacterial aggregate densities was found in diseased samples but no potential pathogens were observed.

  16. Histological observations in the Hawaiian reef coral, Porites compressa, affected by Porites bleaching with tissue loss

    USGS Publications Warehouse

    Sudek, M.; Work, T.M.; Aeby, G.S.; Davy, S.K.

    2012-01-01

    The scleractinian finger coral Porites compressa is affected by the coral disease Porites bleaching with tissue loss (PBTL). This disease initially manifests as bleaching of the coenenchyme (tissue between polyps) while the polyps remain brown with eventual tissue loss and subsequent algal overgrowth of the bare skeleton. Histopathological investigation showed a loss of symbiont and melanin-containing granular cells which was more pronounced in the coenenchyme than the polyps. Cell counts confirmed a 65% reduction in symbiont density. Tissue loss was due to tissue fragmentation and necrosis in affected areas. In addition, a reduction in putative bacterial aggregate densities was found in diseased samples but no potential pathogens were observed.

  17. A Microscale Approach for the Quantitative Detection of PCBs and PAHs in Small Tissue Masses

    DTIC Science & Technology

    2007-10-01

    tissue cleanup methods. 3 - 4 g wet weight tissue Add 200 mL 1:1 hexane/acetone, Extract by Soxhlet Cleanup, Concentrate to 1 mL No further...tissue mass by additional concentration of the final extract volume. Working with lower sample masses or extract volumes is challenging due to...difficulties in monitoring final extract volume, the concomitant concentration of compounds that Report Documentation Page Form ApprovedOMB No. 0704-0188

  18. Tools to assess tissue quality.

    PubMed

    Neumeister, Veronique M

    2014-03-01

    Biospecimen science has recognized the importance of tissue quality for accurate molecular and biomarker analysis and efforts are made to standardize tissue procurement, processing and storage conditions of tissue samples. At the same time the field has emphasized the lack of standardization of processes between different laboratories, the variability inherent in the analytical phase and the lack of control over the pre-analytical phase of tissue processing. The problem extends back into tissue samples in biorepositories, which are often decades old and where documentation about tissue processing might not be available. This review highlights pre-analytical variations in tissue handling, processing, fixation and storage and emphasizes the effects of these variables on nucleic acids and proteins in harvested tissue. Finally current tools for quality control regarding molecular or biomarker analysis are summarized and discussed.

  19. Mouse tissue fixation.

    PubMed

    Cardiff, Robert D; Miller, Claramae H; Munn, Robert J

    2014-05-01

    One of the primary goals of fixation is to stop postmortem changes that degrade the tissue and allow optimal preservation of morphologic and cytological detail as well as nucleic acid integrity. Following death, tissues soon undergo autolysis, and if organisms from the gastrointestinal, urinary, or respiratory tracts are present, their colonization can soon cause putrefaction. Time is of the essence because warmer temperatures accelerate both types of degradation. Placing the tissue into a fixative stops the postmortem changes. Fixatives have their effect on tissue by cross-linking, coagulation, or a combination of both. This article outlines the basic tissue fixation procedure and offers guidance on choosing an appropriate fixative, the timing and duration of fixation, sample storage, and quality issues.

  20. Development and Validation of an Improved PCR Method Using the 23S-5S Intergenic Spacer for Detection of Rickettsiae in Dermacentor variabilis Ticks and Tissue Samples from Humans and Laboratory Animals

    PubMed Central

    Kakumanu, Madhavi L.; Ponnusamy, Loganathan; Sutton, Haley T.; Meshnick, Steven R.; Nicholson, William L.

    2016-01-01

    A novel nested PCR assay was developed to detect Rickettsia spp. in ticks and tissue samples from humans and laboratory animals. Primers were designed for the nested run to amplify a variable region of the 23S-5S intergenic spacer (IGS) of Rickettsia spp. The newly designed primers were evaluated using genomic DNA from 11 Rickettsia species belonging to the spotted fever, typhus, and ancestral groups and, in parallel, compared to other Rickettsia-specific PCR targets (ompA, gltA, and the 17-kDa protein gene). The new 23S-5S IGS nested PCR assay amplified all 11 Rickettsia spp., but the assays employing other PCR targets did not. The novel nested assay was sensitive enough to detect one copy of a cloned 23S-5S IGS fragment from “Candidatus Rickettsia amblyommii.” Subsequently, the detection efficiency of the 23S-5S IGS nested assay was compared to those of the other three assays using genomic DNA extracted from 40 adult Dermacentor variabilis ticks. The nested 23S-5S IGS assay detected Rickettsia DNA in 45% of the ticks, while the amplification rates of the other three assays ranged between 5 and 20%. The novel PCR assay was validated using clinical samples from humans and laboratory animals that were known to be infected with pathogenic species of Rickettsia. The nested 23S-5S IGS PCR assay was coupled with reverse line blot hybridization with species-specific probes for high-throughput detection and simultaneous identification of the species of Rickettsia in the ticks. “Candidatus Rickettsia amblyommii,” R. montanensis, R. felis, and R. bellii were frequently identified species, along with some potentially novel Rickettsia strains that were closely related to R. bellii and R. conorii. PMID:26818674

  1. Development and Validation of an Improved PCR Method Using the 23S-5S Intergenic Spacer for Detection of Rickettsiae in Dermacentor variabilis Ticks and Tissue Samples from Humans and Laboratory Animals.

    PubMed

    Kakumanu, Madhavi L; Ponnusamy, Loganathan; Sutton, Haley T; Meshnick, Steven R; Nicholson, William L; Apperson, Charles S

    2016-04-01

    A novel nested PCR assay was developed to detectRickettsiaspp. in ticks and tissue samples from humans and laboratory animals. Primers were designed for the nested run to amplify a variable region of the 23S-5S intergenic spacer (IGS) ofRickettsiaspp. The newly designed primers were evaluated using genomic DNA from 11Rickettsiaspecies belonging to the spotted fever, typhus, and ancestral groups and, in parallel, compared to otherRickettsia-specific PCR targets (ompA,gltA, and the 17-kDa protein gene). The new 23S-5S IGS nested PCR assay amplified all 11Rickettsiaspp., but the assays employing other PCR targets did not. The novel nested assay was sensitive enough to detect one copy of a cloned 23S-5S IGS fragment from "CandidatusRickettsia amblyommii." Subsequently, the detection efficiency of the 23S-5S IGS nested assay was compared to those of the other three assays using genomic DNA extracted from 40 adultDermacentor variabilisticks. The nested 23S-5S IGS assay detectedRickettsiaDNA in 45% of the ticks, while the amplification rates of the other three assays ranged between 5 and 20%. The novel PCR assay was validated using clinical samples from humans and laboratory animals that were known to be infected with pathogenic species ofRickettsia The nested 23S-5S IGS PCR assay was coupled with reverse line blot hybridization with species-specific probes for high-throughput detection and simultaneous identification of the species ofRickettsiain the ticks. "CandidatusRickettsia amblyommii,"R. montanensis,R. felis, andR. belliiwere frequently identified species, along with some potentially novelRickettsiastrains that were closely related toR. belliiandR. conorii.

  2. Acellular biological tissues containing inherent glycosaminoglycans for loading basic fibroblast growth factor promote angiogenesis and tissue regeneration.

    PubMed

    Lai, Po-Hong; Chang, Yen; Chen, Sung-Ching; Wang, Chung-Chi; Liang, Huang-Chien; Chang, Wei-Chun; Sung, Hsing-Wen

    2006-09-01

    It was found in our previous study that acellular tissues derived from bovine pericardia consist primarily of insoluble collagen, elastin, and tightly bound glycosaminoglycans (GAGs). It is speculated that the inherent GAGs in acellular tissues may serve as a reservoir for loading basic fibroblast growth factor (bFGF) and promote angiogenesis and tissue regeneration. This study was therefore designed to investigate effects of the content of GAGs in acellular bovine pericardia on the binding of bFGF and its release profile in vitro while its stimulation in angiogenesis and tissue regeneration in vivo were evaluated subcutaneously in a rat model. To control the content of GAGs, acellular tissues were treated additionally with hyaluronidase for 1 (Hase-D1), 3 (Hase-D3), or 5 days (Hase-D5). The in vitro results indicated that a higher content of GAGs in the acellular tissue resulted in an increase in bFGF binding and in a more gradual and sustained release of the growth factor. The in vivo results obtained at 1 week postoperatively showed that the density and the depth of neo-vessels infiltrated into the acellular tissue loaded with bFGF (acellular/bFGF) were significantly greater than the other test samples. At 1 month postoperatively, vascularized neo-connective tissues were found to fill the pores within each test sample, particularly for the acellular/bFGF tissue. These results suggested that the sustained release of bFGF from the acellular/ bFGF tissue continued to be effective in enhancing angiogenesis and generation of new tissues. In conclusion, the inherent GAGs present in acellular tissues may be used for binding and sustained release of bFGF to enhance angiogenesis and tissue regeneration.

  3. Tissue types (image)

    MedlinePlus

    There are 4 basic types of tissue: connective tissue, epithelial tissue, muscle tissue, and nervous tissue. Connective tissue supports other tissues and binds them together (bone, blood, and lymph tissues). ...

  4. Tissue engineering of reproductive tissues and organs.

    PubMed

    Atala, Anthony

    2012-07-01

    Regenerative medicine and tissue engineering technology may soon offer new hope for patients with serious injuries and end-stage reproductive organ failure. Scientists are now applying the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that can restore and maintain normal function in diseased and injured reproductive tissues. In addition, the stem cell field is advancing, and new discoveries in this field will lead to new therapeutic strategies. For example, newly discovered types of stem cells have been retrieved from uterine tissues such as amniotic fluid and placental stem cells. The process of therapeutic cloning and the creation of induced pluripotent cells provide still other potential sources of stem cells for cell-based tissue engineering applications. Although stem cells are still in the research phase, some therapies arising from tissue engineering endeavors that make use of autologous adult cells have already entered the clinic. This article discusses these tissue engineering strategies for various organs in the male and female reproductive tract.

  5. [Tissue engineering in reconstructive urology].

    PubMed

    Engel, O; Soave, A; Rink, M; Dahlem, R; Hellwinkel, O; Chun, F K; Fisch, M

    2015-05-01

    The term tissue engineering incorporates various techniques for the production of replacement tissues and organs. In urology tissue engineering offers many promising possibilities for the reconstruction of the urinary tract. Currently, buccal mucosa and urothelial cells are most commonly used for tissue engineering of the urinary tract. Various materials have been tested for their suitability as tissue scaffolds. The ideal scaffold, however, has not yet been found. In addition to material sciences and cell culture methods, surgical techniques play an important role in reconstructive urology for the successful implantation of tissue engineered transplants.

  6. Archaea prevalence in inflamed pulp tissues

    PubMed Central

    Efenberger, Magdalena; Agier, Justyna; Pawłowska, Elżbieta

    2015-01-01

    Archaea have been detected in several ecological niches of the human body such as the large intestine, skin, vagina as well as the oral cavity. At present, archaea are recognized as nonpathogenic microorganisms. However, some data indicate that they may be involved in the etiopathogenesis of several diseases, including intestinal diseases as well as oral diseases: periodontitis, peri-implantitis and endodontitis. In this study, on the basis of 16S rRNA gene sequence analysis, we examined whether archaea might be present in inflamed pulp tissues and contribute to the development of endodontic infection. In comparison, we also determined selected bacterial species associated with endodontitis. We detected archaea in 85% of infected endodontic samples. In addition, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola were present in inflamed pulp tissue samples and Treponema denticola occurred with the highest frequency (70%). Further analysis revealed the presence of methanogenic archaea in analyzed samples. Direct sequencing of archaeal 16S rRNA gene PCR products indicated the occurrence of methanogenic archaea in inflamed pulp tissues; phylogenetically most similar were Methanobrevibacter oralis and Methanobrevibacter smithii. Therefore, our results show that methanogenic archaea are present in inflamed pulp tissues and may participate in the development of endodontic infection. PMID:26557034

  7. Detection of Quiescent Infections with Multiple Elephant Endotheliotropic Herpesviruses (EEHVs), Including EEHV2, EEHV3, EEHV6, and EEHV7, within Lymphoid Lung Nodules or Lung and Spleen Tissue Samples from Five Asymptomatic Adult African Elephants

    PubMed Central

    Zong, Jian-Chao; Heaggans, Sarah Y.; Long, Simon Y.; Latimer, Erin M.; Nofs, Sally A.; Bronson, Ellen; Casares, Miguel; Fouraker, Michael D.; Pearson, Virginia R.; Richman, Laura K.

    2015-01-01

    ABSTRACT More than 80 cases of lethal hemorrhagic disease associated with elephant endotheliotropic herpesviruses (EEHVs) have been identified in young Asian elephants worldwide. Diagnostic PCR tests detected six types of EEHV in blood of elephants with acute disease, although EEHV1A is the predominant pathogenic type. Previously, the presence of herpesvirus virions within benign lung and skin nodules from healthy African elephants led to suggestions that African elephants may be the source of EEHV disease in Asian elephants. Here, we used direct PCR-based DNA sequencing to detect EEHV genomes in necropsy tissue from five healthy adult African elephants. Two large lung nodules collected from culled wild South African elephants contained high levels of either EEHV3 alone or both EEHV2 and EEHV3. Similarly, a euthanized U.S. elephant proved to harbor multiple EEHV types distributed nonuniformly across four small lung nodules, including high levels of EEHV6, lower levels of EEHV3 and EEHV2, and a new GC-rich branch type, EEHV7. Several of the same EEHV types were also detected in random lung and spleen samples from two other elephants. Sanger PCR DNA sequence data comprising 100 kb were obtained from a total of 15 different strains identified, with (except for a few hypervariable genes) the EEHV2, EEHV3, and EEHV6 strains all being closely related to known genotypes from cases of acute disease, whereas the seven loci (4.0 kb) obtained from EEHV7 averaged 18% divergence from their nearest relative, EEHV3. Overall, we conclude that these four EEHV species, but probably not EEHV1, occur commonly as quiescent infections in African elephants. IMPORTANCE Acute hemorrhagic disease characterized by high-level viremia due to infection by members of the Proboscivirus genus threatens the future breeding success of endangered Asian elephants worldwide. Although the genomes of six EEHV types from acute cases have been partially or fully characterized, lethal disease predominantly

  8. X-ray Phase Contrast Imaging of Calcified Tissue and Biomaterial Structure in Bioreactor Engineered Tissues

    SciTech Connect

    Appel, Alyssa A.; Larson, Jeffery C.; Garson, III, Alfred B.; Guan, Huifeng; Zhong, Zhong; Nguyen, Bao-Ngoc; Fisher, John P.; Anastasio, Mark A.; Brey, Eric M.

    2014-11-04

    Tissues engineered in bioreactor systems have been used clinically to replace damaged tissues and organs. In addition, these systems are under continued development for many tissue engineering applications. The ability to quantitatively assess material structure and tissue formation is critical for evaluating bioreactor efficacy and for preimplantation assessment of tissue quality. These techniques allow for the nondestructive and longitudinal monitoring of large engineered tissues within the bioreactor systems and will be essential for the translation of these strategies to viable clinical therapies. X-ray Phase Contrast (XPC) imaging techniques have shown tremendous promise for a number of biomedical applications owing to their ability to provide image contrast based on multiple X-ray properties, including absorption, refraction, and scatter. In this research, mesenchymal stem cell-seeded alginate hydrogels were prepared and cultured under osteogenic conditions in a perfusion bioreactor. The constructs were imaged at various time points using XPC microcomputed tomography (µCT). Imaging was performed with systems using both synchrotron- and tube-based X-ray sources. XPC µCT allowed for simultaneous three-dimensional (3D) quantification of hydrogel size and mineralization, as well as spatial information on hydrogel structure and mineralization. Samples were processed for histological evaluation and XPC showed similar features to histology and quantitative analysis consistent with the histomorphometry. Furthermore, these results provide evidence of the significant potential of techniques based on XPC for noninvasive 3D imaging engineered tissues grown in bioreactors.

  9. Multiphoton cryo microscope with sample temperature control

    NASA Astrophysics Data System (ADS)

    Breunig, H. G.; Uchugonova, A.; König, K.

    2013-02-01

    We present a multiphoton microscope system which combines the advantages of multiphoton imaging with precise control of the sample temperature. The microscope provides online insight in temperature-induced changes and effects in plant tissue and animal cells with subcellular resolution during cooling and thawing processes. Image contrast is based on multiphoton fluorescence intensity or fluorescence lifetime in the range from liquid nitrogen temperature up to +600°C. In addition, micro spectra from the imaged regions can be recorded. We present measurement results from plant leaf samples as well as Chinese hamster ovary cells.

  10. Mechanical Behavior of Tissue Simulants and Soft Tissues Under Extreme Loading Conditions

    NASA Astrophysics Data System (ADS)

    Kalcioglu, Zeynep Ilke

    Recent developments in computer-integrated surgery and in tissue-engineered constructs necessitate advances in experimental and analytical techniques in characterizing properties of mechanically compliant materials such as gels and soft tissues, particularly for small sample volumes. One goal of such developments is to quantitatively predict and mimic tissue deformation due to high rate impact events typical of industrial accidents and ballistic insults. This aim requires advances in mechanical characterization to establish tools and design principles for tissue simulant materials that can recapitulate the mechanical responses of hydrated soft tissues under dynamic contact-loading conditions. Given this motivation, this thesis studies the mechanical properties of compliant synthetic materials developed for tissue scaffold applications and of soft tissues, via modifying an established contact based technique for accurate, small scale characterization under fully hydrated conditions, and addresses some of the challenges in the implementation of this method. Two different engineered material systems composed of physically associating block copolymer gels, and chemically crosslinked networks including a solvent are presented as potential tissue simulants for ballistic applications, and compared directly to soft tissues from murine heart and liver. In addition to conventional quasistatic and dynamic bulk mechanical techniques that study macroscale elastic and viscoelastic properties, new methodologies are developed to study the small scale mechanical response of the aforementioned material systems to concentrated impact loading. The resistance to penetration and the energy dissipative constants are quantified in order to compare the deformation of soft tissues and mechanically optimized simulants, and to identify the underlying mechanisms by which the mechanical response of these tissue simulant candidates are modulated. Finally, given that soft tissues are biphasic in

  11. New addition curing polyimides

    NASA Technical Reports Server (NTRS)

    Frimer, Aryeh A.; Cavano, Paul

    1991-01-01

    In an attempt to improve the thermal-oxidative stability (TOS) of PMR-type polymers, the use of 1,4-phenylenebis (phenylmaleic anhydride) PPMA, was evaluated. Two series of nadic end-capped addition curing polyimides were prepared by imidizing PPMA with either 4,4'-methylene dianiline or p-phenylenediamine. The first resulted in improved solubility and increased resin flow while the latter yielded a compression molded neat resin sample with a T(sub g) of 408 C, close to 70 C higher than PME-15. The performance of these materials in long term weight loss studies was below that of PMR-15, independent of post-cure conditions. These results can be rationalized in terms of the thermal lability of the pendant phenyl groups and the incomplete imidization of the sterically congested PPMA. The preparation of model compounds as well as future research directions are discussed.

  12. Porosity of additive manufacturing parts for process monitoring

    SciTech Connect

    Slotwinski, J. A.; Garboczi, E. J.

    2014-02-18

    Some metal additive manufacturing processes can produce parts with internal porosity, either intentionally (with careful selection of the process parameters) or unintentionally (if the process is not well-controlled.) Material porosity is undesirable for aerospace parts - since porosity could lead to premature failure - and desirable for some biomedical implants, since surface-breaking pores allow for better integration with biological tissue. Changes in a part's porosity during an additive manufacturing build may also be an indication of an undesired change in the process. We are developing an ultrasonic sensor for detecting changes in porosity in metal parts during fabrication on a metal powder bed fusion system, for use as a process monitor. This paper will describe our work to develop an ultrasonic-based sensor for monitoring part porosity during an additive build, including background theory, the development and detailed characterization of reference additive porosity samples, and a potential design for in-situ implementation.

  13. Porosity of additive manufacturing parts for process monitoring

    NASA Astrophysics Data System (ADS)

    Slotwinski, J. A.; Garboczi, E. J.

    2014-02-01

    Some metal additive manufacturing processes can produce parts with internal porosity, either intentionally (with careful selection of the process parameters) or unintentionally (if the process is not well-controlled.) Material porosity is undesirable for aerospace parts - since porosity could lead to premature failure - and desirable for some biomedical implants, since surface-breaking pores allow for better integration with biological tissue. Changes in a part's porosity during an additive manufacturing build may also be an indication of an undesired change in the process. We are developing an ultrasonic sensor for detecting changes in porosity in metal parts during fabrication on a metal powder bed fusion system, for use as a process monitor. This paper will describe our work to develop an ultrasonic-based sensor for monitoring part porosity during an additive build, including background theory, the development and detailed characterization of reference additive porosity samples, and a potential design for in-situ implementation.

  14. Tissue repair

    PubMed Central

    2010-01-01

    As living beings that encounter every kind of traumatic event from paper cut to myocardial infarction, we must possess ways to heal damaged tissues. While some animals are able to regrow complete body parts following injury (such as the earthworm who grows a new head following bisection), humans are sadly incapable of such feats. Our means of recovery following tissue damage consists largely of repair rather than pure regeneration. Thousands of times in our lives, a meticulously scripted but unseen wound healing drama plays, with cells serving as actors, extracellular matrix as the setting and growth factors as the means of communication. This article briefly reviews the cells involved in tissue repair, their signaling and proliferation mechanisms and the function of the extracellular matrix, then presents the actors and script for the three acts of the tissue repair drama. PMID:21220961

  15. Tissue Issues

    ERIC Educational Resources Information Center

    Metz, James

    2016-01-01

    Every day, 27,000 trees are used to make bathroom tissue. Americans use an average of 23.6 rolls per person per year, and more than 7 billion rolls of toilet paper are sold yearly in the United States alone. Perhaps the amount of bathroom tissue used can be reduced by changing the dimensions of the paper or the core. This brief article presents…

  16. Source attribution of agriculture-related deposition by using total nitrogen and δ¹⁵N in epiphytic lichen tissue, bark and deposition water samples in Germany.

    PubMed

    Boltersdorf, Stefanie; Werner, Willy

    2013-06-01

    Compared with physico-chemical deposition measurement methods, lichens are able to identify the long-term overall effects of high N pollution concentrations in the air. In addition, the natural abundances of the stable isotope of N, (15)N, are being widely used in research on N cycling in ecosystems. They can also be used as instruments for source attribution. In this study, epiphytic lichens were tested to determine whether their respective N content and δ(15)N ratios can be used to estimate N deposition rates and to locate various sources of N compounds. Epiphytic lichen and bark samples were collected from around various deposition measurement field stations at different sites in the western part of Germany. The N content of epiphytic lichens reflects the species-specific, agriculture-related circumstances of N deposition at various sites in Germany. At the same time, δ(15)N signatures of the different investigated epiphytic lichen species and bark samples are highly depleted in (15)N under high ammonium deposition. The different surface types of lichens and barks exhibit different concentrations of N and δ(15)N ratios, despite being exposed to similar N deposition rates. The verification of highly negative δ(15)N ratios at sites with local and regional emitters shows that source attribution is possible by comparing different δ(15)N signatures in areas with a wide range of different N deposition types and the corresponding differences in δ(15)N among various source N pools. Especially nitrophytic lichens can support the on-site instrumentation measuring N deposition by qualification and quantification.

  17. Viscoelastic properties of bovine orbital connective tissue and fat: constitutive models.

    PubMed

    Yoo, Lawrence; Gupta, Vijay; Lee, Choongyeop; Kavehpore, Pirouz; Demer, Joseph L

    2011-12-01

    Reported mechanical properties of orbital connective tissue and fat have been too sparse to model strain-stress relationships underlying biomechanical interactions in strabismus. We performed rheological tests to develop a multi-mode upper convected Maxwell (UCM) model of these tissues under shear loading. From 20 fresh bovine orbits, 30 samples of connective tissue were taken from rectus pulley regions and 30 samples of fatty tissues from the posterior orbit. Additional samples were defatted to determine connective tissue weight proportion, which was verified histologically. Mechanical testing in shear employed a triborheometer to perform: strain sweeps at 0.5-2.0 Hz; shear stress relaxation with 1% strain; viscometry at 0.01-0.5 s(-1) strain rate; and shear oscillation at 1% strain. Average connective tissue weight proportion was 98% for predominantly connective tissue and 76% for fatty tissue. Connective tissue specimens reached a long-term relaxation modulus of 668 Pa after 1,500 s, while corresponding values for fatty tissue specimens were 290 Pa and 1,100 s. Shear stress magnitude for connective tissue exceeded that of fatty tissue by five-fold. Based on these data, we developed a multi-mode UCM model with variable viscosities and time constants, and a damped hyperelastic response that accurately described measured properties of both connective and fatty tissues. Model parameters differed significantly between the two tissues. Viscoelastic properties of predominantly connective orbital tissues under shear loading differ markedly from properties of orbital fat, but both are accurately reflected using UCM models. These viscoelastic models will facilitate realistic global modeling of EOM behavior in binocular alignment and strabismus.

  18. Viscoelastic properties of bovine orbital connective tissue and fat: constitutive models

    PubMed Central

    Yoo, Lawrence; Gupta, Vijay; Lee, Choongyeop; Kavehpore, Pirouz

    2012-01-01

    Reported mechanical properties of orbital connective tissue and fat have been too sparse to model strain–stress relationships underlying biomechanical interactions in strabismus. We performed rheological tests to develop a multi-mode upper convected Maxwell (UCM) model of these tissues under shear loading. From 20 fresh bovine orbits, 30 samples of connective tissue were taken from rectus pulley regions and 30 samples of fatty tissues from the posterior orbit. Additional samples were defatted to determine connective tissue weight proportion, which was verified histologically. Mechanical testing in shear employed a triborheometer to perform: strain sweeps at 0.5–2.0 Hz; shear stress relaxation with 1% strain; viscometry at 0.01–0.5 s−1 strain rate; and shear oscillation at 1% strain. Average connective tissue weight proportion was 98% for predominantly connective tissue and 76% for fatty tissue. Connective tissue specimens reached a long-term relaxation modulus of 668 Pa after 1,500 s, while corresponding values for fatty tissue specimens were 290 Pa and 1,100 s. Shear stress magnitude for connective tissue exceeded that of fatty tissue by five-fold. Based on these data, we developed a multimode UCM model with variable viscosities and time constants, and a damped hyperelastic response that accurately described measured properties of both connective and fatty tissues. Model parameters differed significantly between the two tissues. Viscoelastic properties of predominantly connective orbital tissues under shear loading differ markedly from properties of orbital fat, but both are accurately reflected using UCM models. These viscoelastic models will facilitate realistic global modeling of EOM behavior in binocular alignment and strabismus. PMID:21207094

  19. Photo Cleavable Polymers for Tissue Engineering

    NASA Astrophysics Data System (ADS)

    Apostol, Monica

    We have found that P4VP and PMMA thin films can be etched with UVA radiation. Furthermore, we also found that dermal fibroblasts could be cultured successfully on the P4VP polymer, with a doubling time comparable to tissue culture Petri dish standards. Consequently we were able to grow tissue on P4VP substrates which could easily be lifted using UVA radiation. The cells that were removed were then re-plated at a lower density and a series of assays was performed at 3 and 6 days. While only a small amount of damage was discernable at day 3 nearly complete recovery was observed at day 6. The technique was also used to pattern areas within the tissue, where other types of cells could be inserted. In order to demonstrate the technique, a hybrid tissue layer was produced, where the dermal fibroblasts in a circular area at the center of the sample were removed via exposure through a mask. A keratinocyte layer was inserted which adhere to the fibroblast layer forming a tissue with integrated layers of two distinct cell types. We also investigated the effects of coated TiO2 particles on cells exposed to UVC. We found that as expected, cells were adversely affected by exposure to UVC and died even after exposure to as little as 3.5 J/cm 2. Addition of 0.4mg/ml TiO2 particles that were uncoated did not provide protection, and the cells died at the same rate. Addition of 4mg/ml of coated TiO2 on the other hand, did not affect cell viability in the absence of UV light and increased the viability after exposure to UVC radiation. In fact the cells containing the coated particles were indistinguishable for the unexposed control samples even after exposure to as much as 7.1J/cm 2 of UVC.

  20. Cullin 5 Expression in the Rat: Cellular and Tissue Distribution, and Changes in Response to Water Deprivation and Hemorrhagic Shock

    DTIC Science & Technology

    2006-05-31

    rehydration Table 2. cul-5 mRNA ( geometric mean + SEM) of rat tissue 86 following water deprivation and rehydration Chapter 5 Table 1...the arithmetic average was computed as the geometric mean for each tissue sample. The standard error of the geometric mean was computed by...multiplying the geometric mean values for each tissue by the ln of the standard error of the mean [see Casella and Berger, 1990, p. 330, for additional

  1. Capillary sample

    MedlinePlus

    ... repeat the test with blood drawn from a vein. Alternative Names Blood sample - capillary; Fingerstick; Heelstick Images Phenylketonuria test Phenylketonuria test Capillary sample References Garza ...

  2. Tissue Classification

    SciTech Connect

    Robinson, David Gerald

    2015-01-01

    The project began as a e ort to support InLight and Lumidigm. With the sale of the companies to a non-New Mexico entity, the project then focused on supporting a new company Medici Technologies. The Small Business (SB) is attempting to quantify glucose in tissue using a series of short interferometer scans of the nger. Each scan is produced from a novel presentation of the nger to the device. The intent of the project is to identify and, if possible, implement improved methods for classi cation, feature selection, and training to improve the performance of predictive algorithms used for tissue classi cation.

  3. Scaffold-free tissue engineering: organization of the tissue cytoskeleton and its effects on tissue shape.

    PubMed

    Czajka, Caitlin A; Mehesz, Agnes Nagy; Trusk, Thomas C; Yost, Michael J; Drake, Christopher J

    2014-05-01

    Work described herein characterizes tissues formed using scaffold-free, non-adherent systems and investigates their utility in modular approaches to tissue engineering. Immunofluorescence analysis revealed that all tissues formed using scaffold-free, non-adherent systems organize tissue cortical cytoskeletons that appear to be under tension. Tension in these tissues was also evident when modules (spheroids) were used to generate larger tissues. Real-time analysis of spheroid fusion in unconstrained systems illustrated modular motion that is compatible with alterations in tensions, due to the process of disassembly/reassembly of the cortical cytoskeletons required for module fusion. Additionally, tissues generated from modules placed within constrained linear molds, which restrict modular motion, deformed upon release from molds. That tissue deformation is due in full or in part to imbalanced cortical actin cytoskeleton tensions resulting from the constraints imposed by mold systems is suggested from our finding that treatment of forming tissues with Y-27632, a selective inhibitor of ROCK phosphorylation, reduced tissue deformation. Our studies suggest that the deformation of scaffold-free tissues due to tensions mediated via the tissue cortical cytoskeleton represents a major and underappreciated challenge to modular tissue engineering.

  4. A Balanced Tissue Composition Reveals New Metabolic and Gene Expression Markers in Prostate Cancer

    PubMed Central

    Tessem, May-Britt; Bertilsson, Helena; Angelsen, Anders; Bathen, Tone F.; Drabløs, Finn; Rye, Morten Beck

    2016-01-01

    Molecular analysis of patient tissue samples is essential to characterize the in vivo variability in human cancers which are not accessible in cell-lines or animal models. This applies particularly to studies of tumor metabolism. The challenge is, however, the complex mixture of various tissue types within each sample, such as benign epithelium, stroma and cancer tissue, which can introduce systematic biases when cancers are compared to normal samples. In this study we apply a simple strategy to remove such biases using sample selections where the average content of stroma tissue is balanced between the sample groups. The strategy is applied to a prostate cancer patient cohort where data from MR spectroscopy and gene expression have been collected from and integrated on the exact same tissue samples. We reveal in vivo changes in cancer-relevant metabolic pathways which are otherwise hidden in the data due to tissue confounding. In particular, lowered levels of putrescine are connected to increased expression of SRM, reduced levels of citrate are attributed to upregulation of genes promoting fatty acid synthesis, and increased succinate levels coincide with reduced expression of SUCLA2 and SDHD. In addition, the strategy also highlights important metabolic differences between the stroma, epithelium and prostate cancer. These results show that important in vivo metabolic features of cancer can be revealed from patient data only if the heterogeneous tissue composition is properly accounted for in the analysis. PMID:27100877

  5. Biophotonics in diagnosis and modeling of tissue pathologies

    NASA Astrophysics Data System (ADS)

    Serafetinides, A. A.; Makropoulou, M.; Drakaki, E.

    2008-12-01

    Biophotonics techniques are applied to several fields in medicine and biology. The laser based techniques, such as the laser induced fluorescence (LIF) spectroscopy and the optical coherence tomography (OCT), are of particular importance in dermatology, where the laser radiation could be directly applied to the tissue target (e.g. skin). In addition, OCT resolves architectural tissue properties that might be useful as tumour discrimination parameters for skin as well as for ocular non-invasive visualization. Skin and ocular tissues are complex multilayered and inhomogeneous organs with spatially varying optical properties. This fact complicates the quantitative analysis of the fluorescence and/or light scattering spectra, even from the same tissue sample. To overcome this problem, mathematical simulation is applied for the investigation of the human tissue optical properties, in the visible/infrared range of the spectrum, resulting in a better discrimination of several tissue pathologies. In this work, we present i) a general view on biophotonics applications in diagnosis of human diseases, ii) some specific results on laser spectroscopy techniques, as LIF measurements, applied in arterial and skin pathologies and iii) some experimental and theoretical results on ocular OCT measurements. Regarding the LIF spectroscopy, we examined the autofluorescence properties of several human skin samples, excised from humans undergoing biopsy examination. A nitrogen laser was used as an excitation source, emitting at 337 nm (ultraviolet excitation). Histopathology examination of the samples was also performed, after the laser spectroscopy measurements and the results from the spectroscopic and medical analysis were compared, to differentiate malignancies, e.g. basal cell carcinoma tissue (BCC), from normal skin tissue. Regarding the OCT technique, we correlated human data, obtained from patients undergoing OCT examination, with Monte Carlo simulated cornea and retina tissues

  6. Determining the extragalactic extinction law with SALT - II. Additional sample

    NASA Astrophysics Data System (ADS)

    Finkelman, Ido; Brosch, Noah; Kniazev, Alexei Y.; Väisänen, Petri; Buckley, David A. H.; O'Donoghue, Darragh; Gulbis, Amanda; Hashimoto, Yas; Loaring, Nicola; Romero-Colmenero, Encarni; Sefako, Ramotholo

    2010-12-01

    We present new results from an ongoing programme to study the dust extragalactic extinction law in E/S0 galaxies with dust lanes with the Southern African Large Telescope (SALT) during its performance verification phase. The wavelength dependence of the dust extinction for seven galaxies is derived in six spectral bands ranging from the near-ultraviolet atmospheric cut-off to the near-infrared. The derivation of an extinction law is performed by fitting model galaxies to the unextinguished parts of the image in each spectral band, and subtracting from these the actual images. We compare our results with the derived extinction law in the Galaxy and find them to run parallel to the Galactic extinction curve with a mean total-to-selective extinction value of RV = 2.71 +/- 0.43. We use total optical extinction values to estimate the dust mass for each galaxy, compare these with dust masses derived from IRAS measurements, and find them to range from 104 to 107 Msolar. We study the case of the well-known dust-lane galaxy NGC2685 for which Hubble Space Telescope/Wide Field Planetary Camera 2 (HST/WFPC2) data are available to test the dust distribution on different scales. Our results imply a scale-free dust distribution across the dust lanes, at least within ~1arcsec (~60 pc) regions. Based on observations made with the Southern African Large Telescope (SALT). E-mail: ido@wise.tau.ac.il (IF); noah@wise.tau.ac.il (NB); akniazev@saao.ac.za (AYK); petri@saao.ac.za (PV); dibnob@saao.ac.za (DAHB); dod@saao.ac.za (DO); amanda@saao.ac.za (AG); hashimot@ntnu.edu.tw (YH); nsl@saao.ac.za (NL); erc@saao.ac.za (ER-C); rrs@saao.ac.za (RS)

  7. Computed tomography characterisation of additive manufacturing materials.

    PubMed

    Bibb, Richard; Thompson, Darren; Winder, John

    2011-06-01

    Additive manufacturing, covering processes frequently referred to as rapid prototyping and rapid manufacturing, provides new opportunities in the manufacture of highly complex and custom-fitting medical devices and products. Whilst many medical applications of AM have been explored and physical properties of the resulting parts have been studied, the characterisation of AM materials in computed tomography has not been explored. The aim of this study was to determine the CT number of commonly used AM materials. There are many potential applications of the information resulting from this study in the design and manufacture of wearable medical devices, implants, prostheses and medical imaging test phantoms. A selection of 19 AM material samples were CT scanned and the resultant images analysed to ascertain the materials' CT number and appearance in the images. It was found that some AM materials have CT numbers very similar to human tissues, FDM, SLA and SLS produce samples that appear uniform on CT images and that 3D printed materials show a variation in internal structure.

  8. TNT metabolites in animal tissues

    SciTech Connect

    Shugart, L.R.

    1990-01-01

    The overall objectives of this project are: to provide quantitative analytical procedures for the analysis of TNT and at least eight of its metabolites in animal tissues; and to obtain representative samples of tissues from animals from designated Army sites, and to determine the presence or absence of TNT and its metabolites in these samples. The study is divided into two Phases corresponding to the stated overall objectives of the project. 5 figs., 4 tabs.

  9. A time-dependent model for improved biogalvanic tissue characterisation.

    PubMed

    Chandler, J H; Culmer, P R; Jayne, D G; Neville, A

    2015-10-01

    Measurement of the passive electrical resistance of biological tissues through biogalvanic characterisation has been proposed as a simple means of distinguishing healthy from diseased tissue. This method has the potential to provide valuable real-time information when integrated into surgical tools. Characterised tissue resistance values have been shown to be particularly sensitive to external load switching direction and rate, bringing into question the stability and efficacy of the technique. These errors are due to transient variations observed in measurement data that are not accounted for in current electrical models. The presented research proposes the addition of a time-dependent element to the characterisation model to account for losses associated with this transient behaviour. Influence of switching rate has been examined, with the inclusion of transient elements improving the repeatability of the characterised tissue resistance. Application of this model to repeat biogalvanic measurements on a single ex vivo human colon tissue sample with healthy and cancerous (adenocarcinoma) regions showed a statistically significant difference (p < 0.05) between tissue types. In contrast, an insignificant difference (p > 0.05) between tissue types was found when measurements were subjected to the current model, suggesting that the proposed model may allow for improved biogalvanic tissue characterisation.

  10. Ultrasonically Assisted Cutting of Bio-tissues in Microtomy

    NASA Astrophysics Data System (ADS)

    Wang, Dong; Roy, Anish; Silberschmidt, Vadim V.

    Modern-day histology of bio-tissues for supporting stratified medicine diagnoses requires high-precision cutting to ensure high quality extremely thin specimens used in analysis. Additionally, the cutting quality is significantly affected by a wide variety of soft and hard tissues in the samples. This paper deals with development of a next generation of microtome employing introduction of controlled ultrasonic vibration to realise a hybrid cutting process of bio-tissues. The study is based on a combination of advanced experimental and numerical (finite-element) studies of multi-body dynamics of a cutting system. The quality of cut samples produced with the prototype is compared with the state-of-the-art.

  11. Infrared laser ablation sample transfer for MALDI imaging.

    PubMed

    Park, Sung-Gun; Murray, Kermit K

    2012-04-03

    An infrared laser was used to ablate material from tissue sections under ambient conditions for direct collection on a matrix assisted laser desorption ionization (MALDI) target. A 10 μm thick tissue sample was placed on a microscope slide and was mounted tissue-side down between 70 and 450 μm from a second microscope slide. The two slides were mounted on a translation stage, and the tissue was scanned in two dimensions under a focused mid-infrared (IR) laser beam to transfer material to the target slide via ablation. After the material was transferred to the target slide, it was analyzed using MALDI imaging using a tandem time-of-flight mass spectrometer. Images were obtained from peptide standards for initial optimization of the system and from mouse brain tissue sections using deposition either onto a matrix precoated target or with matrix addition after sample transfer and compared with those from standard MALDI mass spectrometry imaging. The spatial resolution of the transferred material is approximately 400 μm. Laser ablation sample transfer provides several new capabilities not possible with conventional MALDI imaging including (1) ambient sampling for MALDI imaging, (2) area to spot concentration of ablated material, (3) collection of material for multiple imaging analyses, and (4) direct collection onto nanostructure assisted laser desorption ionization (NALDI) targets without blotting or ultrathin sections.

  12. NASA Bioreactor tissue culture

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues have reported that initially disc-like specimens tend to become spherical in space, demonstrating that tissues can grow and differentiate into distinct structures in microgravity. The Mir Increment 3 (Sept. 16, 1996 - Jan. 22, 1997) samples were smaller, more spherical, and mechanically weaker than Earth-grown control samples. These results demonstrate the feasibility of microgravity tissue engineering and may have implications for long human space voyages and for treating musculoskeletal disorders on earth. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

  13. Tissue-like phantoms

    DOEpatents

    Frangioni, John V.; De Grand, Alec M.

    2007-10-30

    The invention is based, in part, on the discovery that by combining certain components one can generate a tissue-like phantom that mimics any desired tissue, is simple and inexpensive to prepare, and is stable over many weeks or months. In addition, new multi-modal imaging objects (e.g., beads) can be inserted into the phantoms to mimic tissue pathologies, such as cancer, or merely to serve as calibration standards. These objects can be imaged using one, two, or more (e.g., four) different imaging modalities (e.g., x-ray computed tomography (CT), positron emission tomography (PET), single photon emission computed tomography (SPECT), and near-infrared (NIR) fluorescence) simultaneously.

  14. Venous Sampling

    MedlinePlus

    ... parts of the body, including: Adrenal venous sampling (AVS) , in which blood samples are taken from the ... for a few days before the procedure. For AVS, you will be asked to stop taking certain ...

  15. Tissue proteomics using chemical immobilization and mass spectrometry.

    PubMed

    Shah, Punit; Zhang, Bai; Choi, Caitlin; Yang, Shuang; Zhou, Jianying; Harlan, Robert; Tian, Yuan; Zhang, Zhen; Chan, Daniel W; Zhang, Hui

    2015-01-15

    Proteomics analysis is important for characterizing tissues to gain biological and pathological insights, which could lead to the identification of disease-associated proteins for disease diagnostics or targeted therapy. However, tissues are commonly embedded in optimal cutting temperature medium (OCT) or are formalin-fixed and paraffin-embedded (FFPE) in order to maintain tissue morphology for histology evaluation. Although several tissue proteomic analyses have been performed on FFPE tissues using advanced mass spectrometry (MS) technologies, high-throughput proteomic analysis of OCT-embedded tissues has been difficult due to the interference of OCT in the MS analysis. In addition, molecules other than proteins present in tissues further complicate tissue proteomic analysis. Here, we report the development of a method using chemical immobilization of proteins for peptide extraction (CIPPE). In this method, proteins are chemically immobilized onto a solid support; interferences from tissues and OCT embedding are removed by extensive washing of proteins conjugated on the solid support. Peptides are then released from the solid phase by proteolysis, enabling MS analysis. This method was first validated by eliminating OCT interference from a standard protein, human serum albumin, where all of the unique peaks contributed by OCT contamination were eradicated. Finally, this method was applied for the proteomic analysis of frozen and OCT-embedded tissues using iTRAQ (isobaric tag for relative and absolute quantitation) labeling and two-dimensional liquid chromatography tandem mass spectrometry. The data showed reproducible extraction and quantitation of 10,284 proteins from 3996 protein groups and a minimal impact of OCT embedding on the analysis of the global proteome of the stored tissue samples.

  16. Multispectral fluorescence imaging of human ovarian and Fallopian tube tissue for early stage cancer detection

    NASA Astrophysics Data System (ADS)

    Tate, Tyler; Baggett, Brenda; Rice, Photini; Watson, Jennifer; Orsinger, Gabe; Nymeyer, Ariel C.; Welge, Weston A.; Keenan, Molly; Saboda, Kathylynn; Roe, Denise J.; Hatch, Kenneth; Chambers, Setsuko; Black, John; Utzinger, Urs; Barton, Jennifer

    2015-03-01

    With early detection, five year survival rates for ovarian cancer are over 90%, yet no effective early screening method exists. Emerging consensus suggests that perhaps over 50% of the most lethal form of the disease, high grade serous ovarian cancer, originates in the Fallopian tube. Cancer changes molecular concentrations of various endogenous fluorophores. Using specific excitation wavelengths and emissions bands on a Multispectral Fluorescence Imaging (MFI) system, spatial and spectral data over a wide field of view can be collected from endogenous fluorophores. Wavelength specific reflectance images provide additional information to normalize for tissue geometry and blood absorption. Ratiometric combination of the images may create high contrast between neighboring normal and abnormal tissue. Twenty-six women undergoing oophorectomy or debulking surgery consented the use of surgical discard tissue samples for MFI imaging. Forty-nine pieces of ovarian tissue and thirty-two pieces of Fallopian tube tissue were collected and imaged with excitation wavelengths between 280 nm and 550 nm. After imaging, each tissue sample was fixed, sectioned and HE stained for pathological evaluation. Comparison of mean intensity values between normal, benign, and cancerous tissue demonstrate a general trend of increased fluorescence of benign tissue and decreased fluorescence of cancerous tissue when compared to normal tissue. The predictive capabilities of the mean intensity measurements are tested using multinomial logistic regression and quadratic discriminant analysis. Adaption of the system for in vivo Fallopian tube and ovary endoscopic imaging is possible and is briefly described.

  17. Laser sampling

    NASA Astrophysics Data System (ADS)

    Gorbatenko, A. A.; Revina, E. I.

    2015-10-01

    The review is devoted to the major advances in laser sampling. The advantages and drawbacks of the technique are considered. Specific features of combinations of laser sampling with various instrumental analytical methods, primarily inductively coupled plasma mass spectrometry, are discussed. Examples of practical implementation of hybrid methods involving laser sampling as well as corresponding analytical characteristics are presented. The bibliography includes 78 references.

  18. The Frontiers of Additive Manufacturing

    SciTech Connect

    Grote, Christopher John

    2016-03-03

    Additive manufacturing, more commonly known as 3-D printing, has become a ubiquitous tool in science for its precise control over mechanical design. For additive manufacturing to work, a 3-D structure is split into thin 2D slices, and then different physical properties, such as photo-polymerization or melting, are used to grow the sequential layers. The level of control allows not only for devices to be made with a variety of materials: e.g. plastics, metals, and quantum dots, but to also have finely controlled structures leading to other novel properties. While 3-D printing is widely used by hobbyists for making models, it also has industrial applications in structural engineering, biological tissue scaffolding, customized electric circuitry, fuel cells, security, and more.

  19. Additive Similarity Trees

    ERIC Educational Resources Information Center

    Sattath, Shmuel; Tversky, Amos

    1977-01-01

    Tree representations of similarity data are investigated. Hierarchical clustering is critically examined, and a more general procedure, called the additive tree, is presented. The additive tree representation is then compared to multidimensional scaling. (Author/JKS)

  20. [Periodontitis and tissue regeneration].

    PubMed

    Yamazaki, Kazuhisa

    2005-08-01

    Chronic periodontitis is a destructive disease that affects the supporting structures of the teeth including periodontal ligament, cementum, and alveolar bone. If left untreated, patients may lose multiple teeth and extensive prosthetic treatment will be required. In order to re-engineer lost tooth-supporting tissues, various therapeutic modalities have been used clinically. Periodontal regeneration procedures including guided tissue regeneration have achieved substantial effects. However, there are several issues to be solved. They are highly technique-sensitive, applicable to limited cases which are susceptible to treatment, and supposed to have relatively low predictability. Therefore, it is necessary to develop new approaches to improve the predictability and effectiveness of regenerative therapies for periodontal tissues. Recently, the concept of tissue engineering has been introduced to restore lost tissues more effectively where the biological process of healing is mimicked. To achieve this, integration of three key elements is required: progenitor/stem cells, growth factors and the extracellular matrix scaffold. Although it has been shown that implantation of bone marrow-derived mesenchymal stem cells into periodontal osseous defects induced regeneration of cementum, periodontal ligament and alveolar bone in dogs, further extensive preclinical studies are required. On the other hand, application of growth factors, particularly basic fibroblast growth factor in the treatment of human periodontitis, is promising and is now in clinical trial. Furthermore, the rate of release of growth factor from the scaffold also can profoundly affect the results of tissue engineering strategies and the development of new materials is expected. In addition, as tissue regenerative potential is negatively regulated by aging, the effects of aging have to be clarified to gain complete regeneration.

  1. [Connective tissue and inflammation].

    PubMed

    Jakab, Lajos

    2014-03-23

    The author summarizes the structure of the connective tissues, the increasing motion of the constituents, which determine the role in establishing the structure and function of that. The structure and function of the connective tissue are related to each other in the resting as well as inflammatory states. It is emphasized that cellular events in the connective tissue are part of the defence of the organism, the localisation of the damage and, if possible, the maintenance of restitutio ad integrum. The organism responds to damage with inflammation, the non specific immune response, as well as specific, adaptive immunity. These processes are located in the connective tissue. Sterile and pathogenic inflammation are relatively similar processes, but inevitable differences are present, too. Sialic acids and glycoproteins containing sialic acids have important roles, and the role of Siglecs is also highlighted. Also, similarities and differences in damages caused by pathogens and sterile agents are briefly summarized. In addition, the roles of adhesion molecules linked to each other, and the whole event of inflammatory processes are presented. When considering practical consequences it is stressed that the structure (building up) of the organism and the defending function of inflammation both have fundamental importance. Inflammation has a crucial role in maintaining the integrity and the unimpaired somato-psychological state of the organism. Thus, inflammation serves as a tool of organism identical with the natural immune response, inseparably connected with the specific, adaptive immune response. The main events of the inflammatory processes take place in the connective tissue.

  2. Combined effect of tissue stabilization and protein extraction methods on phosphoprotein analysis.

    PubMed

    Kofanova, Olga A; Fack, Fred; Niclou, Simone P; Betsou, Fay

    2013-06-01

    Preanalytical conditions applied during sample collection and processing can affect the detection or quantification of unstable phosphoprotein biomarkers. We evaluated the consequences of tissue stabilization and protein extraction methods on phosphoprotein analysis. The effects of stabilization techniques (heat stabilization, snap-freezing) and time on the levels of phosphoproteins, including phospho-Akt, p-ERK 1/2, p-IkBα, p-JNK, and p38 MAPK, were evaluated using a BioPlex phosphoprotein assay. Additionally, two different protein extraction protocols, using different extraction buffers (8 M urea buffer, or Bio-Rad buffer without urea) were tested. For snap-frozen samples, protein extraction yields were comparable with the two buffer systems. For heat-stabilized samples, total protein yields were significantly lower following extraction in non-urea buffer. However, the concentrations of specific phosphoproteins were significantly higher in heat-stabilized samples than in the corresponding snap-frozen samples, indicating that this tissue processing method better preserved phosphoproteins. Significant differences were found between the measured phosphoprotein levels in heat-stabilized and snap-frozen tissue, suggesting that alterations occur very rapidly after tissue excision. Our results suggest that heat stabilization can be used as a tissue processing method for subsequent phosphoprotein analyses, but also suggest that the BioPlex phosphoprotein assay could be used as a possible quality control method to assess tissue sample integrity.

  3. Lunar Sample Quarantine & Sample Curation

    NASA Technical Reports Server (NTRS)

    Allton, Judith H.

    2000-01-01

    The main goal of this presentation is to discuss some of the responsibility of the lunar sample quarantine project. The responsibilities are: flying the mission safely, and on schedule, protect the Earth from biohazard, and preserve scientific integrity of samples.

  4. Hormone Receptor Expression Analyses in Neoplastic and Non-Neoplastic Canine Mammary Tissue by a Bead Based Multiplex Branched DNA Assay: A Gene Expression Study in Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Samples

    PubMed Central

    Mohr, Annika; Lüder Ripoli, Florenza; Hammer, Susanne Conradine; Willenbrock, Saskia; Hewicker-Trautwein, Marion; Kiełbowicz, Zdzisław; Murua Escobar, Hugo; Nolte, Ingo

    2016-01-01

    Immunohistochemistry (IHC) is currently considered the method of choice for steroid hormone receptor status evaluation in human breast cancer and, therefore, it is commonly utilized for assessing canine mammary tumors. In case of low hormone receptor expression, IHC is limited and thus is complemented by molecular analyses. In the present study, a multiplex bDNA assay was evaluated as a method for hormone receptor gene expression detection in canine mammary tissues. Estrogen receptor (ESR1), progesterone receptor (PGR), prolactin receptor (PRLR) and growth hormone receptor (GHR) gene expressions were evaluated in neoplastic and non-neoplastic canine mammary tissues. A set of 119 fresh frozen and 180 formalin-fixed, paraffin-embedded (FFPE) was comparatively analyzed and used for assay evaluation. Furthermore, a possible association between the hormone receptor expression in different histological subtypes of canine malignant mammary tumors and the castration status, breed and invasive growth of the tumor were analyzed. The multiplex bDNA assay proved to be more sensitive for fresh frozen specimens. Hormone receptor expression found was significantly decreased in malignant mammary tumors in comparison to non-neoplastic tissue and benign mammary tumors. Among the histological subtypes the lowest gene expression levels of ESR1, PGR and PRLR were found in solid, anaplastic and ductal carcinomas. In summary, the evaluation showed that the measurement of hormone receptors with the multiplex bDNA assay represents a practicable method for obtaining detailed quantitative information about gene expression in canine mammary tissue for future studies. Still, comparison with IHC or quantitative real-time PCR is needed for further validation of the present method. PMID:27649560

  5. Assumed non-persistent environmental chemicals in human adipose tissue; matrix stability and correlation with levels measured in urine and serum.

    PubMed

    Artacho-Cordón, F; Arrebola, J P; Nielsen, O; Hernández, P; Skakkebaek, N E; Fernández, M F; Andersson, A M; Olea, N; Frederiksen, H

    2017-03-22

    The aim of this study was to (1) optimize a method for the measurement of parabens and phenols in adipose tissue, (2) evaluate the stability of chemical residues in adipose tissue samples, and (3) study correlations of these compounds in urine, serum, and adipose tissue. Samples were obtained from adults undergoing trauma surgery. Nine phenols and seven parabens were determined by isotope diluted TurboFlow-LC-MS/MS. The analytical method showed good accuracy and precision. Limits of detection (LOD) for parabens and phenols ranged from 0.05 to 1.83ng/g tissue. Good recovery rates were found, even when biological samples remained defrosted up to 24h. Benzophenone-3 (BP-3; range of values: tissue) and methylparaben (MeP; tissue) were detected in >70% of adipose tissue samples, while bisphenol-A (BPA; tissue) and 2-phenylphenol (2-PP; tissue) were detected in >40% of adipose tissue samples. In general, levels were similar between adipose tissue and serum, while a correlation between adipose tissue and urine was only found for BP-3. In conclusion, adipose tissue samples in this study were found to contain environmental chemicals considered to be non-persistent, whose levels were weakly or not at all correlated with the urine burden. Therefore, adipose tissue may potentially provide additional information to that obtained from other biological matrices. Further investigations are warranted to explore whether adipose tissue might be a suitable matrix for assessment of the consequences for human health of mid/long-term exposure to these chemicals.

  6. Polyimide processing additives

    NASA Technical Reports Server (NTRS)

    Pratt, J. R.; St. Clair, T. L.; Burks, H. D.; Stoakley, D. M.

    1987-01-01

    A method has been found for enhancing the melt flow of thermoplastic polyimides during processing. A high molecular weight 422 copoly(amic acid) or copolyimide was fused with approximately 0.05 to 5 pct by weight of a low molecular weight amic acid or imide additive, and this melt was studied by capillary rheometry. Excellent flow and improved composite properties on graphite resulted from the addition of a PMDA-aniline additive to LARC-TPI. Solution viscosity studies imply that amic acid additives temporarily lower molecular weight and, hence, enlarge the processing window. Thus, compositions containing the additive have a lower melt viscosity for a longer time than those unmodified.

  7. [Food additives and healthiness].

    PubMed

    Heinonen, Marina

    2014-01-01

    Additives are used for improving food structure or preventing its spoilage, for example. Many substances used as additives are also naturally present in food. The safety of additives is evaluated according to commonly agreed principles. If high concentrations of an additive cause adverse health effects for humans, a limit of acceptable daily intake (ADI) is set for it. An additive is a risk only when ADI is exceeded. The healthiness of food is measured on the basis of nutrient density and scientifically proven effects.

  8. Shale JP-4 Additive Evaluation

    DTIC Science & Technology

    1986-10-01

    8217. •% . , ’ ,,,r ,% . -- - ,.-. ’ ’ 4,w% %’. " - ,’ . . . * ’, .* . TABLE OF CONTENTS .4q ,4 . * SECTION PAGE I. INTRODUCTION 1 II. TEST PARAMETERS 2 1...42 PRECEDING PAGE BLANK TABLE OF CONTENTS (CON’T) SECT ION PAGE V. CONCLUSIONS 44 REFERENCES 46 APPENDIX A Drum to Test Sample Relationship 47 APPENDIX...B.O.C.L.E. Results 40 vii LIST OF TABLES TABLE PAGE 1 Antioxidants 3 2 Raw Shale/Petroleum Fuel Properties 10 3 Drum Sample Additive Content 13 4

  9. Application of dried-droplets deposited on pre-cut filter paper disks for quantitative LA-ICP-MS imaging of biologically relevant minor and trace elements in tissue samples.

    PubMed

    Bonta, Maximilian; Hegedus, Balazs; Limbeck, Andreas

    2016-02-18

    In this work, a novel calibration approach for minor and trace element quantification in LA-ICP-MS imaging of biological tissues is presented. Droplets of aqueous standard solutions are deposited onto pre-cut pieces of filter paper, allowed to dry, and sputtered with a thin gold layer for use as pseudo-internal standard. Analysis of the standards using LA-ICP-MS is performed using radial line-scans across the filters. In contrast to conventionally used preparation of matrix-matched tissue standards, the dried-droplet approach offers a variety of advantages: The standards are easy to prepare, no characterization of the standards using acid digestion is required, no handling of biological materials is necessary, and the concentration range, as well the number of investigated analytes is almost unlimited. The proposed quantification method has been verified using homogenized tissue standards with known analyte concentrations before being applied to a human malignant mesothelioma biopsy from a patient who had not received any chemotherapeutic treatment. Elemental distribution images were acquired at a lateral resolution of 40 μm per pixel, limits of detection ranging from 0.1 μg g(-1) (Mn, Ni, Cu, Zn) to 13.2 μg g(-1) (K) were reached.

  10. Tissue Penetration of Antifungal Agents

    PubMed Central

    Felton, Timothy; Troke, Peter F.

    2014-01-01

    SUMMARY Understanding the tissue penetration of systemically administered antifungal agents is critical for a proper appreciation of their antifungal efficacy in animals and humans. Both the time course of an antifungal drug and its absolute concentrations within tissues may differ significantly from those observed in the bloodstream. In addition, tissue concentrations must also be interpreted within the context of the pathogenesis of the various invasive fungal infections, which differ significantly. There are major technical obstacles to the estimation of concentrations of antifungal agents in various tissue subcompartments, yet these agents, even those within the same class, may exhibit markedly different tissue distributions. This review explores these issues and provides a summary of tissue concentrations of 11 currently licensed systemic antifungal agents. It also explores the therapeutic implications of their distribution at various sites of infection. PMID:24396137

  11. Tissue irradiator

    DOEpatents

    Hungate, F.P.; Riemath, W.F.; Bunnell, L.R.

    1975-12-16

    A tissue irradiator is provided for the in-vivo irradiation of body tissue. The irradiator comprises a radiation source material contained and completely encapsulated within vitreous carbon. An embodiment for use as an in- vivo blood irradiator comprises a cylindrical body having an axial bore therethrough. A radioisotope is contained within a first portion of vitreous carbon cylindrically surrounding the axial bore, and a containment portion of vitreous carbon surrounds the radioisotope containing portion, the two portions of vitreous carbon being integrally formed as a single unit. Connecting means are provided at each end of the cylindrical body to permit connections to blood- carrying vessels and to provide for passage of blood through the bore. In a preferred embodiment, the radioisotope is thulium-170 which is present in the irradiator in the form of thulium oxide. A method of producing the preferred blood irradiator is also provided, whereby nonradioactive thulium-169 is dispersed within a polyfurfuryl alcohol resin which is carbonized and fired to form the integral vitreous carbon body and the device is activated by neutron bombardment of the thulium-169