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Vital Staining (vital + staining)
Selected AbstractsIntegration and differentiation of human embryonic stem cells transplanted to the chick embryoDEVELOPMENTAL DYNAMICS, Issue 1 2002Ronald S. Goldstein Abstract Human embryonic stem (ES) cells are pluripotent cells that can differentiate into a large array of cell types and, thus, hold promise for advancing our understanding of human embryology and for contributing to transplantation medicine. In this study, differentiation of human ES cells was examined in vivo by in ovo transplantation to organogenesis-stage embryos. Colonies of human ES cells were grafted into or in place of epithelial-stage somites of chick embryos of 1.5 to 2 days of development. The grafted human ES cells survived in the chick host and were identified by vital staining with carboxyfluorescein diacetate or use of a green fluorescent protein,expressing cells. Histologic analysis showed that human ES cells are easily distinguished from host cells by their larger, more intensely staining nuclei. Some grafted cells differentiated en masse into epithelia, whereas others migrated and mingled with host tissues, including the dorsal root ganglion. Colonies grafted directly adjacent to the host neural tube produced primarily structures with the morphology and molecular characteristics of neural rosettes. These structures contain differentiated neurons as shown by ,-3-tubulin and neurofilament expression in axons and cell bodies. Axons derived from the grafted cells penetrate the host nervous system, and host axons enter the structures derived from the graft. Our results show that human ES cells transplanted in ovo survive, divide, differentiate, and integrate with host tissues and that the host embryonic environment may modulate their differentiation. The chick embryo, therefore, may serve as an accessible and unique experimental system for the study of in vivo development of human ES cells. © 2002 Wiley-Liss, Inc. [source] Chemotaxis of Ralstonia sp.ENVIRONMENTAL MICROBIOLOGY, Issue 10 2006SJ98 towards p -nitrophenol in soil Summary Bioremediation of contaminated sites has been accepted as an efficient and cheaper alternative to physicochemical means of remediation in several cases. Although chemotactic behaviour of many bacteria has been studied earlier and assays have been developed to study bacterial chemotaxis in semi-solid media, this phenomenon has never been demonstrated in soil. For bioremediation application it is important to know whether bacteria actually migrate through the heterogenous soil medium towards a gradient of a particular chemoattractant. In the present study we have successfully demonstrated bacterial chemotaxis of a Ralstonia sp. SJ98 in soil microcosm using qualitative and quantitative plate and tray assays. The migration of bacteria has been established using several methods such as plate counting, vital staining and flow cytometry and slot blot hybridization. A non-chemotactic p- nitrophenol utilizing strain Burkholderia cepacia RKJ200 has been used as negative control. Our work clearly substantiates the hypothesis that chemotactic bacteria may enhance in situ bioremediation of toxic pollutants from soils and sediments. [source] Bacterial population dynamics and community structure in a pharmaceutical manufacturing water supply system determined by real-time PCR and PCR-denaturing gradient gel electrophoresisJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2004M. Kawai Abstract Aims:, To control bacteria in the pharmaceutical water supply system. Methods and Results:, Bacteria were enumerated by conventional culture method and fluorescent vital staining. Activated carbon treatment and storage in a tank provided favourable environments for bacterial growth. The bacterial population of the water in both the post-activated carbon treatment and the tank was analysed by denaturing gradient gel electrophoresis (DGGE) with PCR-amplified 16S rDNA fragments including V6, -7, and -8 regions. The bacterial community structure in activated carbon treated water was stable throughout the year. Several kinds of bacteria such as genus Aquaspirillum and Methylobacterium were found in the water after activated carbon treatment. The bacterial community structure was changed and other bacteria such as mycobacteria were detected after storage. Mycobacteria were quantified in water samples using real-time PCR targeting the 16S rDNA gene. Mycobacteria were also detected in tap water and their number was increased 103,104 -fold higher after storage. Conclusion:, These data suggest the importance of culture-independent methods for quality control of water used in pharmaceutical manufacturing. Significance and Impact of the Study:, Critical steps and specified bacteria that should be controlled in the water supply system were recognized by culture-independent methods. These data will enable effective control of water used in the pharmaceutical industry. [source] Cadmium accumulation and binding characteristics in intact Sertoli/germ cell units, and associated effects on stage-specific functions in vitro: insights from a shark testis modelJOURNAL OF APPLIED TOXICOLOGY, Issue 2 2008Leon M. McClusky Abstract The increased human use of cadmium (Cd) and its increased occurrence in the environment is of concern. The testis is sensitive to Cd because of the steroid-mediated regulation of spermatogenesis, high levels of DNA synthesis and gene transcription, all of which varies in a stage-related manner. Validated techniques (acridine orange vital staining to detect apoptosis and dextran-rhodamine exclusion to assess blood,testis barrier function) were recently developed and the shark testis was proposed as an alternative model for assessing stage-specific functions in living testicular tissue and to study toxicant actions on spermatogenesis. The present paper shows that 109Cd accumulation and binding in vitro was stage-dependent (premeiotic, PrM > meiotic, M > postmeiotic, PoM), rapid and persisted in spermatocysts (intact germ cell/Sertoli cell units) 49 h after washout. In competitive binding analyses of all three spermatocyst stages, Hg, but not Zn, could replace bound 109Cd, suggesting that Cd binding was specific. These findings were associated with a biphasic apoptotic response in the PrM spermatocysts, which was maximal at 10 µm CdCl2 and 1 µm CdCl2 after 2 and 4 days in culture, respectively. Although Cd uptake in PoM cysts was more than 2-fold less than uptake in PrM cysts, the percentage dextran-rhodamine permeant PoM cysts was ,8-fold greater than in controls in the presence of both 10 µm CdCl2 and 30 µm CdCl2 after 4 days culture, indicating that blood,testis barrier function in PoM spermatocysts was compromised. These findings demonstrate that this model has utility for use in screening assays of environmental toxicants. Copyright © 2007 John Wiley & Sons, Ltd. [source] IMPACT OF BLOOD FLOW OCCLUSION ON LIVER NECROSIS FOLLOWING THERMAL ABLATIONANZ JOURNAL OF SURGERY, Issue 1-2 2006Mehrdad Nikfarjam Background: Laser, radiofrequency and microwave are common techniques for local destruction of liver tumours by thermal ablation. The main limitation of thermal ablation treatment is the volume of necrosis that can be achieved. Blood flow occlusion is commonly advocated as an adjunct to thermal ablation to increase the volume of tissue necrosis based on macroscopic and histological assessment of immediate or direct thermal injury. This study examines the impact of blood flow occlusion on direct and indirect laser induced thermal liver injury in a murine model using histochemical methods to assess tissue vitality. Methods: Thermal ablation produced by neodymium yttrium-aluminium-garnet laser (wavelength 1064 nm) was applied to the liver of inbred male CBA strain mice at 2 W for 50 s (100 J). Treatment was performed with and without temporary portal vein and hepatic artery blood flow occlusion. Animals were killed upon completion of the procedure to assess direct thermal injury or at 24, 48 and 72 h to assess the progression of tissue damage. The maximum diameter of necrosis was assessed by vital staining for nicotinamide adenine dinucleotide (NADH) diaphorase. Microvascular changes were assessed by laser Doppler flowmetry, confocal in vivo microscopy and scanning electron microscopy. Results: The direct thermal injury (mean SE) assessed by NADH diaphorase staining was significantly greater following thermal ablation treatment without blood flow occlusion than with blood flow occlusion (3.3 (0.4) mm vs 2.9 (0.3) mm; P = 0.005). Tissue disruption, cracking and vacuolization was more pronounced adjacent to the fibre insertion site in the group treated with thermal ablation combined with blood flow occlusion. There was an equivalent increase in the extent of injury following therapy in both groups that reached a peak at 48 h. The maximum diameter of necrosis in the thermal ablation alone group at 48 h was significantly greater than the thermal ablation combined with blood flow occlusion group (5.8 (0.4) mm vs 5.3 (0.3) mm; P = 0.011). The patterns of microvascular injury were similar in both groups, varying in extent. Conclusion: Temporary blood flow inflow occlusion appears to decrease the extent of initial injury measured by vital staining techniques and does not alter the time sequence of progressive tissue injury following thermal ablation therapy. [source] In vitro activity of dermaseptin S1 derivatives against genital pathogensAPMIS, Issue 9 2010DIANELLA SAVOIA Savoia D, Donalisio M, Civra A, Salvadori S, Guerrini R. In vitro activity of dermaseptin S1 derivatives against genital pathogens. APMIS 2010; 118: 674,80. The aim of this study was to evaluate the biological activity of nine dermaseptin-S1 (DRS-S1) derivatives (synthesized by solid-phase methods and purified) against different pathogens causing genital infections (Trichomonas vaginalis, Herpes simplex virus, Papillomavirus). The in vitro activity on T. vaginalis was determined by counting the protozoon in a hemocytometer after vital staining with trypan blue; antiviral activity of the compounds was tested on monolayers of Vero cells for Herpes simplex virus-1 (GFP) and on 293TT cells for human papillomavirus (HPV-16) pseudovirions (GFP). The cytotoxicity of the derivatives was assessed by evaluating both the hemolytic activity and the effect on Vero and 293TT cells. The DRS-S1 longer peptides demonstrated a superior activity on T. vaginalis but also a certain cytopathic effect. The compounds with 29 amino acids exhibited activity against the two viruses tested at concentrations not toxic to cells. The results obtained show that some of the synthetic peptides assessed have inhibitory activity against the pathogens tested, indicating a potential for the development of new molecules for use as topical microbicides to prevent the sexual transmission of microorganisms. [source] Perturbation of retinoic acid (RA)-mediated limb development suggests a role for diminished RA signaling in the teratogenesis of ethanol,,§BIRTH DEFECTS RESEARCH, Issue 9 2007Corey S. Johnson Abstract BACKGROUND: A proposed mechanism for ethanol teratogenicity entails ethanol-mediated reductions in retinoic acid (RA). This premise was investigated utilizing a mouse model, with limb reduction defects as the teratogenic end point. METHODS: Ethanol, Disulfiram, or BMS-189453 was administered to C57BL/6J mice on the 9th day of pregnancy. Forelimb morphology was assessed on gestation day 18 using Alcian blue and Alizarin red staining. Nile blue sulfate or LysoTracker Red (LTR) vital staining identified cell death in the limb bud. The ability of RA to prevent ethanol-induced cell death was assessed by coadministration followed by laser scanning confocal microscopic examination of LTR-staining. In situ hybridization and qPCR were used to examine gene expression in treated limb buds. RESULTS: Ethanol, Disulfiram, and BMS-189453 resulted in postaxial ectrodactyly, intermediate ectrodactyly, and other digital defects. Excessive Nile blue sulfate staining was evident in the presumptive AER following each of the three exposures. Ethanol-induced LTR staining was prevented by RA supplementation. Both in situ hybridization and qPCR illustrated decreases in Shh and Tbx5 in ethanol-exposed embryos as compared to control. CONCLUSIONS: Contrary to studies of prolonged RA deficiency, acute exposure to functional antagonists of RA results in limb defects that are morphologically similar to those caused by ethanol. The rescue of ethanol-induced cell death by RA and similar changes in Shh transcription further suggest that RA contributes to ethanol-induced limb dysmorphology. Moreover, the repression of key mediators of limb development soon after ethanol exposure adds to the existing knowledge of the pathogenic effects of ethanol. Birth Defects Research (Part A), 2007. © 2007 Wiley-Liss, Inc. [source] 3133: Planar patch-clamping in human corneal endothelial cells: a new tool for clinical application?ACTA OPHTHALMOLOGICA, Issue 2010S MERGLER Purpose Identification of apoptotic or damaged human corneal endothelial cells (HCECs) is limited to morphological evaluation such as phase contrast microscopy and vital staining. The molecular mechanisms of corneal endothelial cell loss are not fully understood. Special investigations in cellular signalling and ion channel research are necessary to elucidate the mechanisms of corneal cell loss. In this context, it is known that this cell loss is often caused by apoptosis in oxidative stress. Methods Automated planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. A particularly successful automated approach is based on planar patch-clamp chips and this is the basis for the technology used here. Routine intracellular or extracellular perfusion opens possibilities for studying the regulation and pharmacology of ion channels. Previously, these studies were available only to highly skilled and dedicated experimenters. Results Notable, definite ion channel activities could be demonstrated by conventional as well as by planar patch-clamp in HCECs for the first time. In particular, temperature-sensing transient receptor potential (TRP)-like non-selective cation channel currents as well as capsaicin-sensitive ion channel currents could be detected. The expression of TRPV1-3 ion channels in HCEC could also be confirmed by RT-PCR, Western blot analysis and fluorescence cell imaging. Conclusion The administration of this novel measuring technology opens new perspectives in the investigation of the physiology of HCEC. The findings may have direct clinical implication (eye banking procedures, keratoplasty). [source] 2432: Histological characteristics of the posterior lid marginACTA OPHTHALMOLOGICA, Issue 2010N KNOP Purpose The structure of the lid margin is insufficiently understood and defined although it is of obvious importance for ocular surface integrity. In particular the histological structure of the tissues of the normal lid margin and their zonal differentiation is partly unclear. Methods The structure and function of the different zones at the posterior lid margin are explained with a focus on dry eye disease, based on the available literature on the lid margin together with own findings on the histology of normal and pathological tissues from the human lid margin. Results The Meibomian glands (MG) that are of particular significance for the integrity of the ocular surface open still within the cornified epidermis. Their obstructive dysfunction (MGD) is a main cause for dry eye disease. The orifice is followed by the mucocutaneous junction (MCJ) that extends from the abrupt termination of the epidermis to the crest of the inner lid border. The physiological vital stainable line of Marx represents its surface and can be used as a diagnostic tool for the location and functionality of the MG orifices and lacrimal puncta. The marginal conjunctiva starts at the crest of the inner lid border and forms a thickened epithelial cushion. This is the point in closest apposition to the globe, represents the zone that wipes the bulbar surface and distributes the thin pre-ocular tear film. It is hence termed as the lid wiper and pathological alterations that result in a vital staining are a sensitive early indicator of dry eye disease. Conclusion The margin of the eyelid is an important but yet underestimated structure for the maintenance of the pre-ocular tear film and of utmost importance for the preservation of ocular surface integrity and for development of dry eye disease. Support DFG KN317/11 [source] 2434: Alterations of the lid wiper zone in ocular surface diseaseACTA OPHTHALMOLOGICA, Issue 2010J NEPP Purpose The marginal part of the tarsal conjunctiva forms a lid wiper structure that wipes the ocular surface and distributes the tear film during the blink. It was attempted to investigate whether this region of the upper and lower lid shows specific changes in several kinds of ocular surface diseases. Methods 44 eyes of patients from the outdoor department of the university eye clinic Vienna were observed by slit lamp investigation of the ocular surface, vital staining with fluorescein and lissamin green. It was focused on evaluation of the tarsal conjunctiva including the lid margin and the wiper. Vessel dilatation, teleangiectatic changes and vital staining were each designed in three stages. The patients suffered from inflammation or affections of the lid, the conjunctiva and the cornea including chemical burns, graft versus host disease and injuries Results Compared to normal eyes dilatation of vessels and staining was observed more in patents with inflammations but although in serious affections like chemical burn or injuries. Even in corneal affections changes of vessels were observed. Conclusion Changes of the wiper can be observed in several diseases of the ocular surface and may be a sign of strain in this region. [source] | |||||||||||||