Detection of apoptotic cells using a synthetic fluorescent sensor for membrane surfaces that contain phosphatidylserine.
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Annexin A5↗
Biomedical subjects
J P Robinson
Publications and source records attributed to J P Robinson.
Automated quantification and reconstruction of collagen matrix from 3D confocal datasets.
The geometrical structure of fibrous extracellular matrix (ECM) impacts on its biological function. In this report, we demonstrate a new algorithm designed to extract quantitative structural information about individual collagen fibres (orientation, length and diameter) from 3D backscattered-light confocal images of collagen gels. The computed quantitative data allowed us to create surface-rendered 3D images of the investigated sample.
Animals↗
The increase of reactive oxygen species and their inhibition in an isolated guinea pig spinal cord compression model.
STUDY DESIGN: In vitro studies using isolated guinea pig spinal cord. OBJECTIVES: To develop an alternative model using isolated guinea pig spinal cord, which can be used to screen antioxidants for in vivo SCI treatment. SETTING: Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana, USA. METHODS: The compression injury was induced by a constant-displacement of 5-s compression of spinal cord using a modified forceps possessing a spacer. Reactive oxygen species (ROS) were evaluated using three distinct methods: fluorescence microscopy, lipid peroxidation assay, and flow cytometry. RESULTS: The injury-mediated ROS increases are comparable with other in vivo studies and consistent with our previous observation using a similar injury model and measured with electrophysiological and anatomical technique. Further, ascorbic acid, hypothermia, or the combination of both significantly suppressed superoxide and lipid peroxidation. The combination treatment was the most effective when compared with ascorbic acid or hypothermia alone. CONCLUSION: This in vitro model has the advantage of replicating some of the in vivo conditions while gaining the ability to control the experimental conditions. This in vitro model is suitable to study the mechanisms of ROS generation and degradation and can also be used to critically evaluate the effective suppressor of ROS in the contents of spinal cord traumatic injury.
Action Potentials↗
Pooled library tissue tags for EST-based gene discovery.
MOTIVATION: In gene discovery projects based on EST sequencing, effective post-sequencing identification methods are important in determining tissue sources of ESTs within pooled cDNA libraries. In the past, such identification efforts have been characterized by higher than necessary failure rates due to the presence of errors within the subsequence containing the oligo tag intended to define the tissue source for each EST. RESULTS: A large-scale EST-based gene discovery program at The University of Iowa has led to the creation of a unique software method named UITagCreator usable in the creation of large sets of synthetic tissue identification tags. The identification tags provide error detection and correction capability and, in conjunction with automated annotation software, result in a substantial improvement in the accurate identification of the tissue source in the presence of sequencing and base-calling errors. These identification rates are favorable, relative to past paradigms. AVAILABILITY: The UITagCreator source code and installation instructions, along with detection software usable in concert with created tag sets, is freely available at http://genome.uiowa.edu/pubsoft/software.html CONTACT: tomc@eng.uiowa.edu
Algorithms↗
Adsorption of avidin on microfabricated surfaces for protein biochip applications.
The adsorption of the protein avidin from hen egg white on patterns of silicon dioxide and platinum surfaces on a microchip and the use of fluorescent microscopy to detect binding of biotin are described. A silicon dioxide microchip was formed using plasma-enhanced chemical vapor deposition while platinum was deposited using radiofrequency sputtering. After cleaning using a plasma arc, the chips were placed into solutions containing avidin or bovine serum albumin. The avidin was adsorbed onto the microchips from phosphate-buffered saline (PBS) or from PBS to which ammonium sulfate had been added. Avidin was also adsorbed onto bovine serum albumin (BSA)-coated surfaces of oxide and platinum. Fluorescence microscopy was used to confirm adsorption of labeled protein, or the binding of fluorescently labeled biotin onto previously adsorbed, unlabeled avidin. When labeled biotin in PBS was presented to avidin adsorbed onto a BSA-coated microchip, the fluorescence signal was significantly higher than for avidin adsorbed onto the biochip alone. The results show that a simple, low-cost adsorption process can deposit active protein onto a chip in an approach that has potential application in the development of protein biochips for the detection of biological species.
Adsorption↗
Outcomes of pain center treatment in Washington State workers' compensation.
BACKGROUND: Injured workers with chronic pain who have failed conventional therapies often receive treatment at pain centers. This study evaluated the effect of pain center treatment on time loss status of Washington State injured workers. The primary hypothesis was that treatment at a pain center would lead to a reduction in the probability of a worker's receiving time loss benefits at a 2-year follow-up. METHODS: A population-based retrospective cohort study was performed on 2,032 Washington State workers' compensation patients who underwent pain center evaluations. Subjects who received pain center treatment were compared to those who were evaluated but not treated with respect to time loss status at 2-year follow-up. RESULTS: Univariate analysis revealed that at 2-year follow-up, 35% of treated subjects were receiving time loss payments vs. 40% of evaluated only subjects (P < 0.05). Subjects who were younger, female, and less chronic were more likely to undergo pain center treatment and were less likely to be on time loss at 2-year follow-up. In multivariate analyses, which statistically controlled baseline differences between the two groups, there was no difference between treated subjects and evaluated only subjects. CONCLUSIONS: There was no evidence that pain center treatment alters 2-year time loss status of already disabled workers.
Adult↗
Principles of confocal microscopy.
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Animals↗
Three-dimensional imaging of extracellular matrix and extracellular matrix-cell interactions.
In summary, noninvasive and nondestructive imaging modalities such as reflection and autofluorescence can readily be used in conjunction with the 3-D optical sectioning capabilities of confocal and multiphoton microscopy to investigate biological processes within living systems. The elimination of specimen fixation and extensive processing reduces the possibility of structural artifacts and facilitates repeat observations within a single sample. Therefore, information representing up to four dimensions (x, y, z, and time) can be readily collected and reconstructed for purposes of visualization and/or quantitative analysis. An advantage of using the techniques described in this chapter is the possibility of performing quantitative measurement of cell size, surface area, volume, depth (in matrix), orientation, receptor density, as well as fluorescence-based indicators of phenotype and function. At present, we are effectively utilizing these techniques to study collagen fibrillogenesis and ECM assembly, structural aspects of ECM-based biomaterials, as well as cell interactions within 3-D matrices (e.g., migration). New insights provided by these techniques regarding ECM and ECM-cell signaling will further the understanding of tissue structure and function and contribute to the development of new and improved strategies for tissue repair, replacement, and maintenance.
Animals↗
Flow cytometric analysis of microorganisms.
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Animals↗
Beyond sexual assessment: lessons learned from couples post radical prostatectomy.
PURPOSE: To share selected experiences of advanced practice nurses (APNs) who implemented a home-based nursing protocol related to psychosexual function for couples following radical surgery for prostate cancer. DATA SOURCES: Selected research-based articles, the PLISSIT Model for sexual rehabilitation counseling, and the authors' experiences. CONCLUSIONS: Five lessons related to communicating about sexuality and intimacy were synthesized from the experience, including examining knowledge and self-awareness regularly, using a structured interview guide to facilitate the process, developing a trusting relationship with the couple, attending to verbal and nonverbal cues, and providing information about the full range of sexual expression. IMPLICATIONS FOR PRACTICE: Include an assessment of sexual health as an integral part of a general health assessment. Patients do not generally volunteer information about their sexual concerns unless the subject is introduced by the APN.
Clinical Protocols↗
Behavioral treatments for post-prostatectomy incontinence.
Despite improvements in surgical approaches to radical prostatectomy, many patients experience moderate to severe urinary incontinence during the first few postoperative weeks. For some patients, leakage continues for several months or years. Urinary incontinence has a significant impact on quality of life in these typically active patients. Surgical interventions are not recommended until after a 6 to 12 month trial of behavioral interventions. Behavioral interventions include supportive care, diet and medication management, pelvic floor rehabilitation, inhibition techniques, and patient education and support. This integrative review of the literature summarizes current knowledge of behavioral interventions for post-prostatectomy urinary incontinence for the primary care and home care clinician. Aspects of care specific to masculine culture are explored. Recommendations for further research include determination of optimal timing and protocols for pelvic floor rehabilitation, best practices for patient and family education, and development of a standard definition of urinary incontinence so the efficacy of treatments can be compared.
Benchmarking↗
Time-lapse confocal reflection microscopy of collagen fibrillogenesis and extracellular matrix assembly in vitro.
The development of the next generation of biomaterials for restoration of tissues and organs (i.e., tissue engineering) requires a better understanding of the extracellular matrix (ECM) and its interaction with cells. Extracellular matrix is a macromolecular assembly of natural biopolymers including collagens, glycosaminoglycans (GAGs), proteoglycans (PGs), and glycoproteins. Interestingly, several ECM components have the ability to form three-dimensional (3D), supramolecular matrices (scaffolds) in vitro by a process of self-directed polymerization, "self-assembly". It has been shown previously that 3D matrices with distinct architectural and biological properties can be formed from either purified type I collagen or a complex mixture of interstitial ECM components derived from intestinal submucosa. Unfortunately, many of the imaging and analysis techniques available to study these matrices either are unable to provide insight into 3D preparations or demand efforts that are often prohibitory to observations of living, dynamic systems. This is the first report on the use of reflection imaging at rapid time intervals combined with laser-scanning confocal microscopy for analysis of structural properties and kinetics of collagen and ECM assembly in 3D. We compared time-lapse confocal reflection microscopy (TL-CRM) with a well-established spectrophotometric method for determining the self-assembly properties of both purified type I collagen and soluble interstitial ECM. While both TL-CRM and spectrophotometric techniques provided insight into the kinetics of the polymerization process, only TL-CRM allowed qualitative and quantitative evaluation of the structural parameters (e.g., fibril diameter) and 3D organization (e.g., fibril density) of component fibrils over time. Matrices formed from the complex mixture of soluble interstitial ECM components showed an increased rate of assembly, decreased opacity, decreased fibril diameter, and increased fibril density compared to that of purified type I collagen. These results suggested that the PG/GAG components of soluble interstitial ECM were affecting the polymerization of the component collagens. Therefore, the effects of purified and complex mixtures of PG/GAG components on the assembly properties of type I collagen and interstitial ECM were evaluated. The data confirmed that the presence of PG/GAG components altered the kinetics and the 3D fibril morphology of assembled matrices. In summary, TL-CRM was demonstrated to be a new and useful technique for analysis of the 3D assembly properties of collagen and other natural biopolymers which requires no specimen fixation and/or staining.
Animals↗
Basal lamina of avian ovarian follicle: influence on morphology of granulosa cells in-vitro.
Experiments were conducted to determine the influence of basal lamina on the morphology of ovarian granulosa cells in vitro. Pure and intact basal lamina was isolated from the large preovulatory follicles of the chicken ovary and designated basal lamina of avian ovarian follicle (BLAOF). Examination of the isolated basal lamina with electron microscope revealed an ultrastructure that is similar to that of basal lamina in the intact ovarian follicle. Pieces of the intact basal lamina were attached to the bottom of 32 mm culture dishes (BLAOF-coated dishes) in which differentiated granulosa cells isolated from the largest preovulatory follicle or undifferentiated granulosa cells isolated from immature small yellow chicken ovarian follicles were cultured; uncoated dishes served as controls. Granulosa cells incubated on intact basal lamina assumed spherical shape, whereas granulosa cells incubated directly on plastic in control dishes became highly flattened. Interestingly, granulosa cells that attached to plastic close to BLAOF (in BLAOF-containing dishes) became rounded. The storage of BLAOF-coated culture dishes at 4 degrees C for 2 years had no apparent effect on its ability of the matrix material to induce changes in granulosa cell shape. Some components of the basal lamina could be solubilized with guanidine-HCl alone (fraction 1; 90-95% of total protein in BLAOF) with the remaining components solubilized with beta-mercaptoethanol containing guanidine-HCl (fraction 2; 5-10% of total protein in BLAOF). Differentiated and undifferentiated chicken granulosa cells became rounded when incubated in fraction 1-pre-coated wells; whereas those incubated directly on plastic in control wells were flattened. Similarly, when fraction 1 of solubilized basal lamina was added as liquid to incubation mixture, it caused both differentiated and undifferentiated granulosa cells to assume spherical shapes. The storage of fraction 1-coated culture dishes at 4 degrees C for 12 or more months had no apparent effect on its ability to influence granulosa cell shape. Fraction 1-induced changes in granulosa cell shape were similar to those observed for complete and intact basal lamina (BLAOF). These findings demonstrate that intact homologous basal lamina (BLAOF) or its solubilized (fluidized) form can induce normal (in vivo) morphology in granulosa cells. It is suggested that BLAOF or its solubilized form can be used to culture cells in experiments designed to examine the influence of the natural basal lamina microenvironment on cellular behavior and function.
Animals↗
Effect of basal lamina on progesterone production by chicken granulosa cells in vitro--influence of follicular development.
Experiments were conducted in vitro to study the regulation of progesterone production in chicken granulosa cells by homologous basal lamina isolated from preovulatory follicles of chicken ovary. The majority of components of the basal lamina (90-95% by weight) were solubilized with guanidine-HCl (and designated fraction 1); the remaining components were solubilized with beta-mercaptoethanol containing guanidine-HCl (and designated fraction 2). The ability of fraction 1 to regulate progesterone production in granulosa cells obtained from the largest (F(1), mature), third largest (F(3), growing), fifth to seventh largest (F(5-7), growing) follicles and a pool of small yellow follicles (SYF, immature) of chicken ovary was assessed. Granulosa cells isolated from SYF follicles were in the least differentiated (undifferentiated) and those obtained from F(1) follicles were in the most differentiated state. The ability of fraction 1 to regulate progesterone production by chicken granulosa cells was influenced both by the state of cell differentiation and the form of the matrix material (whether solid or liquid). When fraction 1 was added as liquid to the incubation mixture, it promoted progesterone production by granulosa cells at all stages of differentiation; however, it caused a greater relative increase in the amount of progesterone produced by undifferentiated (SYF) and differentiating (F(3)) granulosa cells than by differentiated (F(1)) ones. In the presence of the liquid-form of fraction 1, luteinizing hormone (LH) stimulated progesterone production in differentiated (F(1)) and differentiating (F(5-7)) granulosa cells. Similarly, follicle-stimulating hormone (FSH) stimulated progesterone production by differentiating (F(3)) and undifferentiated (SYF) granulosa cells in the presence of the liquid-form of fraction 1 protein. In culture wells that had been pre-coated with fraction 1 (solid-form), progesterone production by less differentiated (SYF, F(5-7)) granulosa cells was enhanced, whereas progesterone production by differentiated (F(1)) cells was reduced. The solid-form of fraction 1 augmented LH-stimulated progesterone production by less differentiated (F(5-7)) granulosa cells however, it attenuated LH-induced progesterone production in differentiated (F(1)) cells. FSH-promoted progesterone production in granulosa cells from immature follicles (SYF) was augmented by solid-form of fraction 1 whereas the effect of FSH on cells obtained from older follicle (F(3)) was suppressed by solid-form of fraction 1. In experiments in which gonadotropin action was attenuated by solid-form of fraction 1, the amount of progesterone produced in the presence of maximally inhibiting concentrations of fraction 1 protein was greater than control values (no fraction 1, no gonadotropin). These results show that basal lamina of the ovarian follicle can regulate progesterone production by granulosa cells. The data demonstrate that the interactions between the components of basal lamina and LH or FSH on granulosa cell function were dependent on the stage of follicular development and were influenced by the form of the matrix material. It is concluded that the basal lamina of the chicken ovarian follicle is biologically active and regulates granulosa cell function.
Animals↗
Managing urinary incontinence following radical prostatectomy.
New diagnoses of prostate cancer more than tripled between 1990 and 1996, largely because of improved methods of detection and heightened public awareness. Radical prostatectomy is often undertaken in men with prostate cancer who are expected to live at least 10 more years and have tumors confined to the prostate gland. Because of high 10-year survival rates, the demand for radical prostatectomy has increased steadily during the past decade and continues to climb as men pursue a cure and attempt to maximize survival. Survival benefits aside, however, radical prostatectomy carries a significant risk of urinary incontinence, which can dramatically impair quality of life. Despite medicine's steadfast pursuit of continence-preserving approaches to radical prostatectomy, nurses are currently challenged to provide care for a growing cohort of patients with postprostatectomy incontinence. This article reviews the clinical presentation and pathophysiology of postprostatectomy incontinence, including assessment and treatment options.
Humans↗
Studies on the mechanism of diarrhoea induced by Escherichia coli heat-stable enterotoxin (STa) in newborn calves.
Enterotoxigenic Escherichia coli (ETEC) produces a heat-stable enterotoxin (STa) that binds to and activates a putative intestinal receptor, guanylate cyclase, causing an increase in the intracellular levels of cyclic guanosine monophosphate (cGMP). Using flow cytometry and 125I-STa binding assays, we studied the distribution of STa-receptors on enterocytes isolated from different segments of the newborn calf's intestinal tract. We also investigated the effect of STa on the intracellular levels of cGMP and ion transport to the intestinal lumen. More STa-receptors were found on enterocytes prepared from the ileum than on enterocytes obtained from the other segments of the intestinal tract. Guanylate cyclase activity was higher in the ileum of STa-challenged calves than in the ileum of control calves. No changes were observed in the guanylate cyclase activity of the other intestinal segments of the STa-challenged and control calves. Na+ levels, as measured by atomic absorption spectroscopy, were significantly increased in the luminal contents of the ileum of STa-challenged calves, whereas serum Cl- levels were significantly lower in the STa-challenged calves than in control calves. This study supports previous observations on the role of guanylate cyclase in the initiation of STa-induced secretory diarrhoea and suggests that Na+/Cl- coupling may be the major mechanism for the loss of ions in the diarrhoeal response that is mostly induced in the ileum of newborn calves.
Animals↗
Rapid purification of transfected porcine muscle cells.
A detailed methodology is described for fluorescence-activated cell sorting (FACS) of porcine muscle cells that have been transfected to express green fluorescent protein (GFP). Cells are liberated from porcine skeletal muscle and primary cultures are transfected with DNA encoding GFP. Primary cultures are subjected to immunocytochemistry using a primary muscle-specific monoclonal antibody followed by incubation with a phycoerythrin-conjugated second antibody. Transfected myoblasts are sorted from fibroblasts using forward angle light scatter and ninety degree light scatter, phycoerythrin fluorescence, and GFP fluorescence. These procedures allow for isolation of genetically- engineered porcine muscle cells more rapidly than traditional clonal selection procedures. Consequently, FACS provides porcine myoblast populations that retain the majority of their replicative capacity and are not contaminated with non-myogenic cells.
Animals↗
Managing urinary incontinence in the nursing home: residents' perspectives.
The purpose of this study was to develop a substantive theory describing and explaining cognitive, psychosocial, and behavioural aspects of living with urinary incontinence in a nursing home. Using grounded theory methodology, data were obtained from interviews, participant observation, and minutes from meetings of resident advisory councils, and analysed concurrently using the constant comparative method. Three nursing homes located in the metropolitan area of a major American city served as sites for the study. Ethical approval was obtained from the administrator of each facility and institutional review board of a major university. Ten residents served as primary participants, consenting to provide one audiotaped formal interview, subsequent opportunities to observe and discuss their methods of bladder control, and access to their health care records. Another 33 residents from these facilities served as secondary participants, providing opportunities for observation and informal interview when their bladder control needs, issues, or methods were visible to the investigator and seemed relevant to the evolving theory. Data were collected over a period of 17 months. Findings suggest that nursing home residents with actual or potential urinary incontinence engage in a process called managing urinary incontinence. Managing urinary incontinence occurs in the context of believing that it is an inevitable part of ageing and seeking to protect physical, psychological, and social integrity. Managing urinary incontinence is a dynamic process with consequences that are influenced by the degree to which six specific strategies are used with satisfaction by the resident and supported by staff and significant others. Implications for nursing practice and research are discussed.
Aged↗