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Development of Tectal Connectivity across Metamorphosis in the Bullfrog (Rana catesbeiana)
Horowitz, Seth S.
Simmons, Andrea Megela
Brain, Behavior and Evolution
In the bullfrog (Rana catesbeiana), the process of metamorphosis culminates in the appearance of new visual and visuomotor behaviors reflective of the emergence of binocular vision and visually-guided prey capture behaviors as the animal transitions to life on land. Using several different neuroanatomical tracers, we examined the substrates that may underlie these behavioral changes by tracing the afferent and efferent connectivity of the midbrain optic tectum across metamorphic development. Intratectal, tectotoral, tectotegmental, tectobulbar, and tecto-thalamic tracts exhibit similar trajectories of neurobiotin fiber label across the developmental span from early larval tadpoles to adults. Developmental variability was apparent primarily in intensity and distribution of cell and puncta label in target nuclei. Combined injections of cholera toxin subunit β and Phaseolus vulgaris leucoagglutinin consistently label cell bodies, puncta, or fiber segments bilaterally in midbrain targets including the pretectal gray, laminar nucleus of the torus semicircularis, and the nucleus of the medial longitudinal fasciculus. Developmentally stable label was observed bilaterally in medullary targets including the medial vestibular nucleus, lateral vestibular nucleus, and reticular gray, and in forebrain targets including the posterior and ventromedial nuclei of the thalamus. The nucleus isthmi, cerebellum, lateral line nuclei, medial septum, ventral striatum, and medial pallium show more developmentally variable patterns of connectivity. Our results suggest that even during larval development, the optic tectum contains substrates for integration of visual with auditory, vestibular, and somatosensory cues, as well as for guidance of motivated behaviors.
Medial septum; Metamorphosis; Midbrain; Multisensory convergence; Nucleus isthmi; Optic tectum; Tadpole; Thalamus
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Spatial distribution and cellular composition of adult brain proliferative zones in the teleost, Gymnotus omarorum
Olivera-Pasilio, Valentina
Peterson, Daniel A.
Castelló, María E.
Frontiers in Neuroanatomy
Proliferation of stem/progenitor cells during development provides for the generation of mature cell types in the CNS. While adult brain proliferation is highly restricted in the mammals, it is widespread in teleosts. The extent of adult neural proliferation in the weakly electric fish, Gymnotus omarorum has not yet been described. To address this, we used double thymidine analog pulse-chase labeling of proliferating cells to identify brain proliferation zones, characterize their cellular composition, and analyze the fate of newborn cells in adult G. omarorum. Short thymidine analog chase periods revealed the ubiquitous distribution of adult brain proliferation, similar to other teleosts, particularly Apteronotus leptorhynchus. Proliferating cells were abundant at the ventricular-subventricular lining of the ventricular-cisternal system, adjacent to the telencephalic subpallium, the diencephalic preoptic region and hypothalamus, and the mesencephalic tectum opticum and torus semicircularis. Extraventricular proliferation zones, located distant from the ventricular-cisternal system surface, were found in all divisions of the rombencephalic cerebellum. We also report a new adult proliferation zone at the caudal-lateral border of the electrosensory lateral line lobe. All proliferation zones showed a heterogeneous cellular composition. The use of short (24 h) and long (30 day) chase periods revealed abundant fast cycling cells (potentially intermediate amplifiers), sparse slow cycling (potentially stem) cells, cells that appear to have entered a quiescent state, and cells that might correspond to migrating newborn neural cells. Their abundance and migration distance differed among proliferation zones: greater numbers and longer range and/or pace of migrating cells were associated with subpallial and cerebellar proliferation zones.
thymidine analogs; adult cell proliferation; weakly electric fish; electrosensory; teleosts
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Plasticity of Auditory Medullary-Midbrain Connectivity across Metamorphic Development in the Bullfrog, Rana catesbeiana
Horowitz, Seth S.
Chapman, Judith A.
Simmons, Andrea Megela
Brain, Behavior and Evolution
On the basis of patterns of anterograde, retrograde, and bi-directional transport of tracers from both the superior olivary nucleus (SON) and the torus semicircularis (TS), we report anatomical changes in brainstem connectivity across metamorphic development in the bullfrog, Rana catesbeiana. In early and late stages of larval development (Gosner stages 25–37), anterograde or bi-directional tracers injected into the SON produce terminal/fiber label in the contralateral SON and in the ipsilateral TS. Between stages 38–41 (deaf period), only sparse or no terminal/fiber label is visible in these target nuclei. During metamorphic climax (stages 42–46), terminal/fiber label reappears in both the contralateral SON and in the ipsilateral TS, and now also in the contralateral TS. Injections of retrograde tracers into the SON fail to label cell bodies in the ipsilateral TS in deaf period animals, mirroring the previously-reported failure of retrograde transport from the TS to the ipsilateral SON during this developmental time. Bilateral cell body label emerges in the dorsal medullary nucleus and the lateral vestibular nucleus bilaterally as a result of SON transport during the late larval period, while cell body label in the contralateral TS emerges during climax. At all larval stages, injections into the SON produce anterograde and retrograde label in the medial vestibular nucleus bilaterally. These data show anatomical stability in some pathways and plasticity in others during larval development, with the most dramatic changes occurring during the deaf period and metamorphic climax. Animals in metamorphic climax show patterns of connectivity similar to that of froglets and adults, indicating the maturation during climax of central anatomical substrates for hearing in air.
Tadpoles; Anurans; Vestibular nucleus complex; Dorsal medullary nucleus; Superior olivary complex; Torus semicircularis; Lipophilic dyes; PHA-L; Cholera toxin; Metamorphosis
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Determination of key aspects of precursor cell proliferation, cell cycle length and kinetics in the adult mouse subgranular zone
Mandyam, Chitra D.
Harburg, Gwyndolen C.
Eisch, Amelia J.
Neurogenesis studies on the adult mouse hippocampal subgranular zone (SGZ) typically report increases or decreases in proliferation. However, key information is lacking about these proliferating SGZ precursors, from the fundamental - what dose of bromodeoxyuridine (BrdU) is appropriate for labeling all S phase cells? - to the detailed - what are the kinetics of BrdU-labeled cells and their progeny? To address these questions, adult C57BL/6J mice were injected with BrdU and BrdU-immunoreactive (IR) cells were quantified. Initial experiments with a range of BrdU doses (25-500 mg/kg) suggested that 150 mg/kg labels all actively dividing precursors in the mouse SGZ. Experiments using a saturating dose of BrdU suggested BrdU bioavailability is less than 15 minutes, notably shorter than in the developing mouse brain. We next explored precursor division and maturation by tracking the number of BrdU-IR cells and colabeling of BrdU with other cell cycle proteins from 15 min to 30 days after BrdU. We found that BrdU and the G2/M phase protein pHisH3 maximally colocalized 8 hr after BrdU, indicating that the mouse SGZ precursor cell cycle length is 14 hr. In addition, triple labeling with BrdU and PCNA and Ki-67 showed that BrdU-IR precursors and/or their progeny express these endogenous cell cycle proteins up to 4 days after BrdU injection. However, the proportion of BrdU/Ki-67-IR cells declined at a greater rate than the proportion of BrdU/PCNA-IR cells. This suggests that PCNA protein is detectable long after cell cycle exit, and that reliance on PCNA may overestimate the length of time a cell remains in the cell cycle. These findings will be critical for future studies examining the regulation of SGZ precursor kinetics in adult mice, and hopefully will encourage the field to move beyond counting BrdU-IR cells to a more mechanistic analysis of adult neurogenesis.
BrdU; PCNA; pHisH3; Ki-67; mitosis; neurogenesis
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Regional distribution and migration of proliferating cell populations in the adult brain of Hyla cinerea (Anura, Amphibia)
M.Almli, Lynn
Wilczynski, Walter
Brain research
We examined the distribution of adult cell proliferation throughout the brain of an anuran amphibian using 5-bromo-2′-deoxyuridine (BrdU). BrdU, a thymidine analog, is a commonly used cellular marker that is incorporated into actively dividing progenitor cells. Adult green treefrogs, Hyla cinerea, received injections of BrdU and were sacrificed 2 hours, 2 days, 2 weeks, or 30 days later. Immunohistochemistry revealed BrdU-immunopositive (BrdU+) cells to be distributed in ventricular zones throughout the brain. The heaviest concentrations of cells were located in the telencephalon, primarily in the ventrolateral region of the lateral ventricles, and the ventricles of olfactory bulbs. Numerous BrdU+ cells were located around the preoptic and hypothalamic recesses and few around the third ventricle in the diencephalon. Proceeding caudally towards the midbrain, there was a marked decrease in BrdU-labeling and few BrdU+ cells were found in the hindbrain. Consistent with previous studies in ectothermic vertebrates, BrdU+ cells were found predominantly in the ventricular zone (VZ) and immediately adjacent to the VZ; at later time points (i.e., 30 days), the cells appeared to have migrated into parenchymal regions. The extent of cellular proliferation in anurans is similar to that of fishes and reptiles and thus is more widespread compared to mammals.
amphibian; BrdU; cell proliferation; cell migration; ventricular zone
Related Articles
Thymidine analog methods for studies of adult neurogenesis are not equally sensitive
Leuner, Benedetta
Glasper, Erica R.
Gould, Elizabeth
The Journal of comparative neurology
Adult neurogenesis is often studied by labeling new cells with the thymidine analog bromodeoxyuridine (BrdU) and using immunohistochemical methods for their visualization. Using this approach, considerable variability has been reported in the number of new cells produced in the dentate gyrus of adult rodents. We examined whether immunohistochemical methods, including BrdU antibodies from different vendors (Vector, BD, Roche, Dako, Novocastra, Accurate) and DNA denaturation pretreatments, alter the quantitative and qualitative patterns of BrdU labeling. We also compared the sensitivity and specificity of BrdU with two other thymidine analogs, iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU). We found that the number of BrdU-labeled cells in the dentate gyrus of adult rats was dependent on the BrdU antibody used but was unrelated to differences in antibody penetration. Even at a higher concentration, some antibodies stained fewer cells (Vector, Novocastra). A sensitive BrdU antibody (BD) was specific for dividing cells; all BrdU-labeled cells stained for Ki67, an endogenous marker of cell proliferation. We also observed that DNA denaturation pretreatments affected the number of BrdU-labeled cells and staining intensity for a marker of neuronal differentiation, NeuN. Finally, we found that IdU and CldU, when used at molarities comparable to those that label the maximal number of cells with BrdU, are less sensitive. These data suggest that antibody and thymidine analog selection, as well as the staining procedure employed, can affect the number of newly generated neurons detected in the adult brain thus providing a potential explanation for some of the variability in the adult neurogenesis literature.
BrdU; IdU; CldU; hippocampus; dentate gyrus; subventricular zone
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Estrogen Regulation of Cell Proliferation and Distribution of Estrogen Receptor-α in the Brains of Adult Female Prairie and Meadow Voles
The Journal of comparative neurology
Adult female prairie (Microtus ochrogaster) and meadow (M. pennsylvanicus) voles were compared to examine neural cell proliferation and the effects of estrogen manipulation on cell proliferation in the amygdala, ventromedial hypothalamus (VMH), and dentate gyrus of the hippocampus (DG). Unlike prior studies, our study focused on the amygdala and VMH, because they are involved in social behaviors and may underlie behavioral differences between the species. Meadow voles had a higher density of cells labeled with the cell proliferation marker 5-bromo-2′-deoxyuridine (BrdU) in the amygdala and DG than did prairie voles. Treatment with estradiol benzoate (EB) for 3 days increased the density of BrdU-labeled cells in the amygdala, particularly in the posterior cortical (pCorA) and medial (pMeA) nuclei, in meadow, but not prairie, voles. Furthermore, the majority of the BrdU-labeled cells in the pCorA and pMeA displayed either a neuronal or a glial progenitor phenotype, but no species or treatment differences were found in the percentage of neuronal or glial progenitor cells. To understand better estrogen's effects on adult neurogenesis, we also examined estrogen receptor-α (ERα) distribution. Meadow voles had more ERα-labeled cells in the pCorA and VMH, but not in the pMeA or DG, than did prairie voles. In addition, more than one-half of the BrdU-labeled cells in the amygdala of both species coexpressed ERα labeling. Together, these data indicate that estrogen alters cell proliferation in a species- and region-specific manner, and some of these effects may lie in the specific localization of estrogen receptors in the adult vole brain.
neurogenesis; ERα; amygdala; hypothalamus; progenitor
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Immunochemical Detection and Isolation of DNA from Metabolically Active Bacteria
Urbach, Ena
Vergin, Kevin L.
Giovannoni, Stephen J.
Applied and Environmental Microbiology
Most techniques used to assay the growth of microbes in natural communities provide no information on the relationship between microbial productivity and community structure. To identify actively growing bacteria, we adapted a technique from immunocytochemistry to detect and selectively isolate DNA from bacteria incorporating bromodeoxyuridine (BrdU), a thymidine analog. In addition, we developed an immunocytochemical protocol to visualize BrdU-labeled microbial cells. Cultured bacteria and natural populations of aquatic bacterioplankton were pulse-labeled with exogenously supplied BrdU. Incorporation of BrdU into microbial DNA was demonstrated in DNA dot blots probed with anti-BrdU monoclonal antibodies and either peroxidase- or Texas red-conjugated secondary antibodies. BrdU-containing DNA was physically separated from unlabeled DNA by using antibody-coated paramagnetic beads, and the identities of bacteria contributing to both purified, BrdU-containing fractions and unfractionated, starting-material DNAs were determined by length heterogeneity PCR (LH-PCR) analysis. BrdU-containing DNA purified from a mixture of DNAs from labeled and unlabeled cultures showed >90-fold enrichment for the labeled bacterial taxon. The LH-PCR profile for BrdU-containing DNA from a labeled, natural microbial community differed from the profile for the community as a whole, demonstrating that BrdU was incorporated by a taxonomic subset of the community. Immunocytochemical detection of cells with BrdU-labeled DNA was accomplished by in situ probing with anti-BrdU monoclonal antibodies and Texas red-labeled secondary antibodies. Using this suite of techniques, microbial cells incorporating BrdU into their newly synthesized DNA can be quantified and the identities of these actively growing cells can be compared to the composition of the microbial community as a whole. Since not all strains tested could incorporate BrdU, these methods may be most useful when used to gain an understanding of the activities of specific species in the context of their microbial community.
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Immortalized, premalignant epithelial cell populations contain long-lived, label-retaining cells that asymmetrically divide and retain their template DNA
Bussard, Karen M
Boulanger, Corinne A
Kittrell, Frances S
Behbod, Fariba
Medina, Daniel
Smith, Gilbert H
Breast Cancer Research : BCR
During selective segregation of DNA, a cell asymmetrically divides and retains its template DNA. Asymmetric division yields daughter cells whose genome reflects that of the parents, simultaneously protecting the parental cell from genetic errors that may occur during DNA replication. We hypothesized that long-lived epithelial cells are present in immortal, premalignant cell populations, undergo asymmetric division, retain their template DNA strands, and cycle both during allometric growth and during pregnancy.
The glands of 3-week-old immune-competent Balb/C female mice were used intact or cleared of host epithelium and implanted with ductal-limited, lobule-limited, or alveolar-ductal progenitor cells derived from COMMA-D1 pre-malignant epithelial cells. 5-Bromo-2-deoxyuridine (5-BrdU) was administered to identify those cells that retain their template DNA. Nulliparous mice were then either injected with [3H]-thymidine (3H-TdR) to distinguish 5-BrdU label-retaining cells that enter the cell cycle and euthanized, or mated, injected with 3H-TdR, and euthanized at various days after coitus. Sections were stained for estrogen receptor-α (ER-α) or progesterone receptor (PR) with immunohistochemistry. Cells labeled with both 5-BrdU and 3H-TdR were indicative of label-retaining epithelial cells (LRECs).
Cells that retained a 5-BrdU label and cells labeled with [3H]-thymidine were found in all mice and were typically detected along the branching epithelium of mature mouse mammary glands. Cells containing double-labeled nuclei (LRECs) were found in the intact mammary glands of both pregnant and nulliparous mice, and in mammary glands implanted with premalignant cells. Double-labeled cells (3H-TdR/5-BrdU) represent a small portion of cells in the mammary gland that cycle and retain their template DNA (5-BrdU). Some label-retaining cells were also ER-α or PR positive. LRECs distributed their second label (3H-TdR) to daughter cells, and this effect persisted during pregnancy. LRECs, and small focal hyperplasia, were found in all immortalized premalignant mammary-implant groups.
The results indicate that a subpopulation of long-lived, label-retaining epithelial cells (LRECs) is present in immortal premalignant cell populations. These LRECs persist during pregnancy, retain their original DNA, and a small percentage express ER-α and PR. We speculate that LRECs in premalignant hyperplasia represent the long-lived (memory) cells that maintain these populations indefinitely.
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The fate of Müller's glia following experimental retinal detachment: nuclear migration, cell division, and subretinal glial scar formation
Lewis, Geoffrey P.
Chapin, Ethan A.
Luna, Gabriel
Linberg, Kenneth A.
Fisher, Steven K.
Molecular Vision
To study the fate of Müller's glia following experimental retinal detachment, using a "pulse/chase" paradigm of bromodeoxyuridine (BrdU) labeling for the purpose of understanding the role of Müller cell division in subretinal scar formation.
Experimental retinal detachments were created in pigmented rabbit eyes, and 3 days later 10 µg of BrdU was injected intravitreally. The retinas were harvested 4 h after the BrdU was administered (i.e., day 3) or on days 4, 7, and 21 post detachment. The tissue was fixed, embedded in agarose, and sectioned at 100 µm. The sections were labeled with various combinations of probes, including anti-vimentin and anti-S100 (as markers for Müller cells), anti-BrdU, anti-phosphohistone H3 (to identify mitotic cells), and the isolectin B4 (to identify macrophages and microglia). Images were captured using an Olympus Fluoview 500 confocal microscope. To aid in our understanding of how Müller cell nuclei undergo cell division, two additional procedures were used: 1) electron microscopy of normal cat and rabbit retinas and 2) a new method using 5-fluorouracil and subsequent anti-BrdU labeling to detect all Müller cell nuclei, using confocal imaging.
Three days after detachment, anti-vimentin labeled all Müller cells, some of which were also labeled with anti-BrdU. On day 4, many of the anti-BrdU-labeled Müller cell nuclei appeared in columns with one labeled nucleus in the inner nuclear layer and another directly sclerad to it in the outer nuclear layer. By day 7, most anti-BrdU-labeled nuclei were observed in subretinal scars. At 3 weeks, some anti-BrdU-labeled nuclei that remained within the retina did not express vimentin or S100. Anti-phosphohistone H3-labeled (i.e., mitotic) cells, some of which were also labeled with anti-BrdU, were only observed in the outer nuclear layer on day 4, and these nuclei were surrounded by an accumulation of vimentin filaments. Isolectin B4-labeled microglia and macrophages also incorporated BrdU and were observed throughout the retina and in subretinal scars during all times of detachment. Electron microscopy and immunofluorescence labeling of the 5-fluorouracil-injected eyes revealed the presence of a unique structural relationship between Müller cell nuclei and intermediate filament proteins.
Following retinal detachment, many Müller cell nuclei initially migrate to the outer retina, undergo mitosis, and eventually reside in subretinal glial scars, suggesting a possible link between the early division of Müller cells and the process of subretinal gliosis. In addition, a subpopulation of anti-BrdU-labeled cells, presumably once Müller cells, appears to stop expressing well accepted Müller cell marker proteins, suggesting a potential dedifferentiation of some of these cells over time. Additionally, Müller cell nuclei may use intermediate filaments as a "track" for migration into the outer retina and later as an important component of cell division by the accumulation of vimentin filaments around the mitotic nuclei.
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Proliferation dynamics of germinative zone cells in the intact and excitotoxically lesioned postnatal rat brain
Faiz, Maryam
Acarin, Laia
Castellano, Bernardo
Gonzalez, Berta
BMC Neuroscience
The forebrain subventricular zone (SVZ)-olfactory bulb pathway and hippocampal subgranular zone (SGZ) generate neurons into adulthood in the mammalian brain. Neurogenesis increases after injury to the adult brain, but few studies examine the effect of injury on neural and glial precursors in the postnatal brain. To characterize the spatio-temporal dynamics of cell proliferation in the germinative zones, this study utilized a model of postnatal damage induced by NMDA injection in the right sensorimotor cortex at postnatal day 9.
Dividing cell populations were labeled with 5-Bromodeoxyuridine (BrdU) in the intact and damaged postnatal brain. Identity of proliferating cells was determined by double immunolabeling with nestin, GFAP, NeuN and tomato lectin (TL).
In the control brain, grouped BrdU+ cells were observed in the Rostral Migratory Stream (RMS), SVZ and SGZ. Maximal proliferation was seen at P12, persisted until P23 and diminished by P49. After injury, a striking reduction in the number of BrdU+ cells was observed in the ipsilateral SVZ from 10 hours (58% decrease) until 14 days post-lesion (88% decrease). In contrast, an increase in grouped BrdU+ cells was seen in the striatum adjacent to the depleted SVZ. Significantly reduced numbers of BrdU+ cells were also seen in the RMS until 3 days post-lesion. No changes were noted in the SGZ. Both in controls and lesioned hemispheres, BrdU+ cells located in the germinal zones were mostly nestin positive and negative for GFAP, NeuN, and TL. In the SVZ area lining the ventricle, BrdU+/nestin+ cells were mainly located between TL+ ependyma and parenchymal GFAP+ astrocytes. After excitotoxicity, a decrease in the number and orientation of GFAP/nestin+ prolongations leaving the SVZ to the cortex, corpus callosum and striatum was noted until 5 days post-lesion.
Postnatal excitotoxic injury differentially affects proliferating cells in the germinative zones: no change is observed in the dentate gyrus whereas excitotoxicity causes a significant decrease in proliferating cells in the SVZ and RMS. Depletion of BrdU+ cells in the postnatal SVZ and RMS differs from previous studies after adult brain injury and may affect the SVZ-RMS migration and is suggestive of progenitor recruitment to injured areas.
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Patterns of bromodeoxyuridine incorporation and neuropeptide immunoreactivity during arm regeneration in the starfish Asterias rubens
Moss, C.
Philosophical Transactions of the Royal Society B: Biological Sciences
Regeneration of the arm of the starfish, Asterias rubens (L.) (Echinodermata: Asteroidea) was examined using two preparations. The first involved regeneration of the entire arm tip and its associated sensory structures and the second examined regeneration of a small section of radial nerve cord in the mid-arm region. Cell cycle activity was investigated by incorporation of the thymidine analogue, bromodeoxyuridine (BrdU). Details of neuroanatomy were obtained by immunocytochemistry (ICC) using an antiserum to the recently isolated starfish neuropeptide, GFNSALMFamide (S1). BrdU labelling indicated that initial events occur by morphallaxis, with cell cycle activity first apparent after formation of a wound epidermis. As regeneration proceeded, BrdU immunoreactive (IR) nuclei revealed cell cycle activity in cells at the distal ends of the radial nerve cord epidermis, in the coelomic epithelium, the perihaemal and water vascular canal epithelia, and in the forming tube feet of both preparations. By varying the time between BrdU pulses and tissue fixation, the possible migration or differentiation of labelled cells was investigated. Neuropeptide ICC indicated the extension of S1-IR nerve fibres into the regenerating area, soon after initial wound healing processes were complete. These fibres were varicose and disorganized in appearance, when compared to the normal pattern of S1-IR in the radial nerve. S1-IR was also observed in cell bodies, which reappeared in the reforming optic cushion and radial nerve at later stages of regeneration. Double labelling studies with anti-BrdU and anti-S1 showed no co-localization in these cell bodies, in all the stages examined. It appeared that S1-IR cells were not undergoing, and had not recently undergone, cell cycle activity. It cannot be confirmed whether S1-IR neurons were derived from proliferating cells of epithelial origin, or from transdifferentiation of epithelial cells, although the former mechanism is suggested. Differentiation of the regenerating structures to replace cells such as S1-containing neurons, is thought to involve cell cycle activity and differentiation of epithelial cells in the epidermal tissue, possibly in association with certain types of coelomocytes which move into the regenerating area.
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Comparison of labelling by bromodeoxyuridine, MIB-1, and proliferating cell nuclear antigen in gastric mucosal biopsy specimens.
Lynch, D A
Clarke, A M
Jackson, P
Axon, A T
Dixon, M F
Quirke, P
Journal of Clinical Pathology
AIMS--To compare proliferating cell nuclear antigen (PCNA) and MIB-1 with bromodeoxyuridine (BrdU) pulse labelling, a specific marker of cell proliferation, in endoscopic gastric biopsy specimens. METHODS--Twenty four biopsy specimens were obtained from 12 patients: 10 antral and eight body specimens were suitable. Each specimen was routinely processed and stained with haematoxylin and eosin. A modified Giemsa stain was used to detect the presence of Helicobacter pylori. Sections of the specimens were labelled with BrdU, MIB-1, and PC10. Gastric mucosa specimens were divided into three zones. The numbers of positively staining nuclei for 500 epithelial cell nuclei were counted in each zone and expressed as a percentage. RESULTS--The proportion of PCNA positive cells (range 0-90%) was much greater in all specimens (10 antrum, eight body). BrdU positive cells were virtually all confined to zone 2 (0-17% cells in this zone were positive) (zone 1 = surface and gastric pit, zone 2 = isthmus, zone 3 = gland base), while PCNA positive cells were present in all three zones (1 = 23-90%, 2 = 43-90%, 3 = 0-74%). Spearman's rank coefficient correlation of 0.57 confirmed that the percentage of positively staining cells varied in the same direction for both PCNA and BrdU (p < 0.001). PCNA, however, was overexpressed in all zones of the gastric epithelium compared with BrdU. In 38 biopsy specimens from 19 patients, of which 14 antrum and 11 body were suitable, the proportion of MIB-1 positive cells (0-59%) was greater than BrdU in most. As with BrdU labelling, the MIB-1 positive cells were confined to zone 2 (zone 1 = 1-11%); zone 2 = 21-59%; zone 3 = 0-13%) and the coefficient correlation for MIB-1 and BrdU was 0.63 (p < 0.001). CONCLUSIONS--MIB-1 accurately reflects the S-phase fraction in gastric mucosa, determined by BrdU labelling in conventionally processed gastric biopsy material. Caution is needed in the interpretation of PCNA labelling detected by PC10, which should not be accepted uncritically as a marker of cell proliferation in paraffin wax embedded material.
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Adult hippocampal neurogenesis and c-Fos induction during escalation of voluntary wheel running in C57BL/6J mice
Clark, Peter J.
Kohman, Rachel A.
Miller, Daniel S.
Bhattacharya, Tushar K.
Haferkamp, Erik H.
Rhodes, Justin S.
Behavioural brain research
Voluntary wheel running activates dentate gyrus granule neurons and increases adult hippocampal neurogenesis. Average daily running distance typically increases over a period of 3 weeks in rodents. Whether neurogenesis and cell activation are greater at the peak of running as compared to the initial escalation period is not known. Therefore, adult C57BL/6J male mice received 5 days of BrdU injections, at the same age, to label dividing cells during the onset of wheel access or after 21 days during peak levels of running or in sedentary conditions. Mice were sampled either 24 hours or 25 days after the last BrdU injection to measure cell proliferation and survival, respectively. Immunohistochemistry was performed on brain sections to identify the numbers of proliferating BrdU labeled cells, and new neurons (BrdU/NeuN co-labeled) in the dentate gyrus. Ki67 was used as an additional mitotic marker. The induction of c-Fos was used to identify neurons activated from running. Mice ran approximately half as far during the first 5 days as compared to after 21 days. Running increased Ki67 cells at the onset but after 21 days levels were similar to sedentary. Numbers of BrdU cells were similar in all groups 24 hours after the final injection. However, after 25 days, running approximately doubled the survival of new neurons born either at the onset or peak of running. These changes co-varied with c-Fos expression. We conclude that sustained running maintains a stable rate of neurogenesis above sedentary via activity-dependent increases in differentiation and survival, not proliferation, of progenitor cells in the C57BL/6J model.
exercise; wheel running; c-Fos; adult hippocampal neurogenesis; granule cell activation; C57BL/6J
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The cell cycle–apoptosis connection revisited in the adult brain
Bauer, Sylvian
Patterson, Paul H.
The Journal of Cell Biology
Adult neurogenesis is studied in vivo using thymidine analogues such as bromodeoxyuridine (BrdU) to label DNA synthesis during the S phase of the cell cycle. However, BrdU may also label DNA synthesis events not directly related to cell proliferation, such as DNA repair and/or abortive reentry into the cell cycle, which can occur as part of an apoptotic process in postmitotic neurons. In this study, we used three well-characterized models of injury-induced neuronal apoptosis and the combined visualization of cell birth (BrdU labeling) and death (Tdt-mediated dUTP-biotin nick end labeling) to investigate the specificity of BrdU incorporation in the adult mouse brain in vivo. We present evidence that BrdU is not significantly incorporated during DNA repair and that labeling is not detected in vulnerable or dying postmitotic neurons, even when a high dose of BrdU is directly infused into the brain. These findings have important implications for a controversy surrounding adult neurogenesis: the connection between cell cycle reactivation and apoptosis of terminally differentiated neurons.
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Reduced Proliferation in the Adult Mouse Subventricular Zone Increases Survival of Olfactory Bulb Interneurons
Sui, Yi
Horne, Malcolm K.
Stanić, Davor
PLoS ONE
Neurogenesis in the adult brain is largely restricted to the subependymal zone (SVZ) of the lateral ventricle, olfactory bulb (OB) and the dentate subgranular zone, and survival of adult-born cells in the OB is influenced by factors including sensory experience. We examined, in mice, whether survival of adult-born cells is also regulated by the rate of precursor proliferation in the SVZ. Precursor proliferation was decreased by depleting the SVZ of dopamine after lesioning dopamine neurons in the substantia nigra compacta with 6-hydroxydopamine. Subsequently, we examined the effect of reduced SVZ proliferation on the generation, migration and survival of neuroblasts and mature adult-born cells in the SVZ, rostral migratory stream (RMS) and OB. Proliferating cells in the SVZ, measured by 5-bromo-2-deoxyuridine (BrdU) injected 2 hours prior to death or by immunoreactivity against Ki67, were reduced by 47% or 36%, respectively, 7 days after dopamine depletion, and by 29% or 31% 42 days after dopamine depletion, compared to sham-treated animals. Neuroblast generation in the SVZ and their migration along the RMS were not affected, neither 7 nor 42 days after the 6-hydroxydopamine injection, since the number of doublecortin-immunoreactive neuroblasts in the SVZ and RMS, as well as the number of neuronal nuclei-immunoreactive cells in the OB, were stable compared to control. However, survival analysis 15 days after 6-hydroxydopamine and 6 days after BrdU injections showed that the number of BrdU+ cells in the SVZ was 70% higher. Also, 42 days after 6-hydroxydopamine and 30 days after BrdU injections, we found an 82% increase in co-labeled BrdU+/γ-aminobutyric acid-immunoreactive cell bodies in the granular cell layer, while double-labeled BrdU+/tyrosine hydroxylase-immunoreactive cell bodies in the glomerular layer increased by 148%. We conclude that the number of OB interneurons following reduced SVZ proliferation is maintained through an increased survival of adult-born precursor cells, neuroblasts and interneurons.
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Neurodistribution of Androgen Receptor Immunoreactivity in the Male Frog, Rana esculenta
Guerriero, G.
Prins, G. S.
Birch, L.
Ciarcia, G.
Annals of the New York Academy of Sciences
Sexual behavior in vertebrates depends on the cyclic release of steroids and their binding to the brain receptors. Previously, we demonstrated the presence of specific binding of 3H-testosterone and staining with PG-21 in the brain of the adult male frog, Rana esculenta. Here, we report our further receptor characterization using an anti–androgen receptor antiserum, PG-21, and the androgen site of action in frog brain. Nuclei, which contained cells labeled for the androgen receptor (AR), were mainly identified in the olfactory bulbs, preoptic-septal region, infundibulum, amygdala, thalamus, tectum, torus semicircularis, and medulla. The neuroanatomical AR staining appears similar to that in other lower vertebrates.
androgen receptor; amphibian; brain; PG-21
Related Articles
A mechanistic model for bromodeoxyuridine dilution naturally explains labelling data of self-renewing T cell populations
Ganusov, Vitaly V.
De Boer, Rob J.
Journal of the Royal Society Interface
Bromodeoxyuridine (BrdU) is widely used in immunology to detect cell division, and several mathematical models have been proposed to estimate proliferation and death rates of lymphocytes from BrdU labelling and de-labelling curves. One problem in interpreting BrdU data is explaining the de-labelling curves. Because shortly after label withdrawal, BrdU+ cells are expected to divide into BrdU+ daughter cells, one would expect a flat down-slope. As for many cell types, the fraction of BrdU+ cells decreases during de-labelling, previous mathematical models had to make debatable assumptions to be able to account for the data. We develop a mechanistic model tracking the number of divisions that each cell has undergone in the presence and absence of BrdU, and allow cells to accumulate and dilute their BrdU content. From the same mechanistic model, one can naturally derive expressions for the mean BrdU content (MBC) of all cells, or the MBC of the BrdU+ subset, which is related to the mean fluorescence intensity of BrdU that can be measured in experiments. The model is extended to include subpopulations with different rates of division and death (i.e. kinetic heterogeneity). We fit the extended model to previously published BrdU data from memory T lymphocytes in simian immunodeficiency virus-infected and uninfected macaques, and find that the model describes the data with at least the same quality as previous models. Because the same model predicts a modest decline in the MBC of BrdU+ cells, which is consistent with experimental observations, BrdU dilution seems a natural explanation for the observed down-slopes in self-renewing populations.
cell turnover; mathematical model; BrdU
Related Articles
Effects of Chronic Furosemide Treatment and Age on Cell Division in the Adult Gerbil Inner Ear
Lang, H.
Schulte, B. A.
Schmiedt, R. A.
JARO: Journal of the Association for Research in Otolaryngology
Atrophy of the stria vascularis and spiral ligament and an associated decrease in the endocochlear potential (EP) are significant factors in age-related hearing loss (presbyacusis). To model this EP decrease, furosemide was delivered into the round-window niche of young adult gerbils by osmotic pump for seven days, chronically reducing the EP by 30–40 mV. Compound action potential (CAP) thresholds were correspondingly reduced by 30–40 dB SPL at high frequencies. Two weeks after withdrawal of furosemide, the treated ears showed an EP recovery of up to 20–30 mV along with a similar recovery of CAP thresholds. The influence of cell division on furosemide-induced and age-related decline of the EP was examined using a mitotic tracer, bromodeoxyuridine (BrdU). Cell proliferation was examined in three groups: young control, furosemide-treated, and aged cochleas. Sections immunostained for BrdU were bleached with H2O2 to eliminate ambiguities with melanin pigment in the inner ear. Cell types positively labeled for BrdU in all three groups included Schwann cells in Rosenthal's canal; glial cells in the osseous spiral lamina; fibrocytes in the limbus, sacculus, and spiral ligament (SL); epithelial cells in Reissner's and round-window membranes; intermediate cells in the stria vascularis; and vascular endothelial cells. Quantitative analysis showed that the mean number of BrdU-positive (BrdU+) intermediate cells in the stria did not differ significantly among the three groups. In contrast, there was a significant increase of BrdU + fibrocytes in the SL of furosemide-treated animals as compared to the young control group. Moreover, there was a significant decrease in labeled fibrocytes in the aged versus the young ears, particularly among the type II and type IV subtypes. The results suggest that the increased fibrocyte turnover in the SL after furosemide treatment may be related to the recovery of EP and CAP thresholds, supporting the hypothesis that fibrocyte proliferation may be essential for maintaining the EP and cochlear function in normal and damaged cochleas. Moreover, the decreased turnover of SL fibrocytes with age may be a contributing factor underlying the lateral wall pathology and consequent EP loss that often accompanies presbyacusis.
cochlea; furosemide; BrdU; aging; fibrocytes; spiral ligament; endocochlear potential
Related Articles
Second-order projection from the posterior lateral line in the early zebrafish brain
Fame, Ryann M
Brajon, Carole
Ghysen, Alain
Neural Development
Mechanosensory information gathered by hair cells of the fish lateral-line system is collected by sensory neurons and sent to the ipsilateral hindbrain. The information is then conveyed to other brain structures through a second-order projection. In the adult, part of the second-order projection extends to the contralateral hindbrain, while another part connects to a midbrain structure, the torus semicircularis.
In this paper we examine the second-order projection from the posterior lateral-line system in late embryonic/early larval zebrafish. At four days after fertilization the synaptic field of the sensory neurons can be accurately targeted, allowing a very reproducible labeling of second-order neurons. We show that second-order projections are highly stereotyped, that they vary according to rhombomeric identity, and that they are almost completely lateralized. We also show that the projections extend not only to the contralateral hindbrain and torus semicircularis but to many other brain centers as well, including gaze- and posture-controlling nuclei in the midbrain, and presumptive thalamic nuclei.
We propose that the extensive connectivity observed in early brain development reveals a basic scaffold common to most vertebrates, from which different subsets are later reinforced in various vertebrate groups. The large repertoire of projection targets provides a promising system to study the genetic encoding of this differential projection capacity.
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Hypoxia–Ischemia Induces DNA Synthesis without Cell Proliferation in Dying Neurons in Adult Rodent Brain
Kuan, Chia-Yi
Schloemer, Aryn J.
Lu, Aigang
Burns, Kevin A.
Weng, Wei-Lan
Williams, Michael T.
Strauss, Kenneth I.
Vorhees, Charles V.
Flavell, Richard A.
Davis, Roger J.
Sharp, Frank R.
Rakic, Pasko
The Journal of neuroscience : the official journal of the Society for Neuroscience
Recent studies suggest that postmitotic neurons can reenter the cell cycle as a prelude to apoptosis after brain injury. However, most dying neurons do not pass the G1/S-phase checkpoint to resume DNA synthesis. The specific factors that trigger abortive DNA synthesis are not characterized. Here we show that the combination of hypoxia and ischemia induces adult rodent neurons to resume DNA synthesis as indicated by incorporation of bromodeoxyuridine (BrdU) and expression of G1/S-phase cell cycle transition markers. After hypoxia–ischemia, the majority of BrdU- and neuronal nuclei (NeuN)-immunoreactive cells are also terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL)-stained, suggesting that they undergo apoptosis. BrdU+ neurons, labeled shortly after hypoxia–ischemia, persist for >5 d but eventually disappear by 28 d. Before disappearing, these BrdU+/NeuN+/TUNEL+neurons express the proliferating cell marker Ki67, lose the G1-phase cyclin-dependent kinase (CDK) inhibitors p16INK4 and p27Kip1 and show induction of the late G1/S-phase CDK2 activity and phosphorylation of the retinoblastoma protein. This contrasts to kainic acid excitotoxicity and traumatic brain injury, which produce TUNEL-positive neurons without evidence of DNA synthesis or G1/S-phase cell cycle transition. These findings suggest that hypoxia–ischemia triggers neurons to reenter the cell cycle and resume apoptosis-associated DNA synthesis in brain. Our data also suggest that the demonstration of neurogenesis after brain injury requires not only BrdU uptake and mature neuronal markers but also evidence showing absence of apoptotic markers. Manipulating the aberrant apoptosis-associated DNA synthesis that occurs with hypoxia–ischemia and perhaps neurodegenerative diseases could promote neuronal survival and neurogenesis.
neurogenesis; apoptosis; cell cycle; BrdU; ischemia; hypoxia
Related Articles
Association between Toll-Like Receptor 4 Expression and Neural Stem Cell Proliferation in the Hippocampus Following Traumatic Brain Injury in Mice
Ye, Yuqin
Xu, Hongyu
Zhang, Xin
Li, Ze
Jia, Yanfeng
He, Xiaosheng
Huang, Jason H.
International Journal of Molecular Sciences
Whether or how neural stem cells (NSCs) respond to toll-like receptor 4 (TLR4) in an inflammatory environment caused by traumatic brain injury (TBI) has not been understood. In the present study, association between TLR4 expression and NSCs proliferation in the hippocampus was investigated in a mouse model of TBI using controlled cortical impact (CCI). Hippocampal proliferating cells were labeled with the thymidine analog 5-bromo-2-deoxyuridine (BrdU). In order to identify NSCs, the proliferating cells were further co-labeled with BrdU/sex determination region of Y chromosome related high mobility group box gene 2 (SOX2). Morphological observation on the expression of BrdU, SOX2, and TLR4 in the hippocampus was performed by inmmunofluorescence (IF). Relative quantification of TLR4 expression at the protein and mRNA level was performed using Western blotting and real-time polymerase chain reaction (PCR). It was observed that BrdU+/SOX2+ cells accounted for 95.80% ± 7.91% among BrdU+ cells; several BrdU+ cells and SOX2+ cells in the hippocampus were also TLR4-positive post injury, and that BrdU+ cell numbers, together with TLR4 expression at either protein or mRNA level, increased significantly in TBI mice over 1, 3, 7, 14, and 21 days survivals and changed in a similar temporal pattern with a peak at 3 day post-injury. These results indicate that hippocampal proliferating cells (suggestive of NSCs) expressed TLR4, and that there was a potential association between increased expression of TLR4 and the proliferation of NSCs post TBI. It is concluded that hippocampal TLR4 may play a potential role in endogenous neurogenesis after TBI.
toll-like receptor 4 (TLR4); neural stem cells (NSCs); hippocampus; traumatic brain injury (TBI); mice
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Proliferating cell nuclear antigen: a marker for hepatocellular proliferation in rodents.
Eldrige, S R
Butterworth, B E
Goldsworthy, T L
Environmental Health Perspectives
1993;101(Suppl 5):211-218.
Two different markers for quantitating cell proliferation were evaluated in livers of control and chemically treated mice and rats. Proliferating cell nuclear antigen (PCNA), an endogenous cell replication marker, and bromodeoxyuridine (BrdU), an exogenously administered DNA precursor label, were detected in formalin-fixed, paraffin-embedded tissues using immunohistochemical techniques. The percentage of cells in S phase (labeling indexes, LI) evaluated as PCNA- or BrdU-positive hepatocellular nuclei was compared in recut tissue sections from animals given BrdU by a single IP injection 2 hr before killing the animals. Ten-week-old male B6C3F1 mice and F344 rats were exposed to known mitogenic hepatocarcinogens, Wy-14,643 (WY) in the diet at 0.1% for 2 days or 1,4-dichlorobenzene (DCB) in corn oil by gavage for 2 days (600 mg/kg/day in mice; 300 mg/kg/day in rats). In mice, PCNA and BrdU hepatocyte LI were similar in control, WY-treated, and DCB-treated animals. In rats, PCNA and BrdU gave similar LI in controls and Wy-treated animals. Although PCNA LI was statistically lower than BrdU LI in DCB-treated rats, both PCNA and BrdU LI for DCB-treated rats was increased over LI in control rats. Different patterns of PCNA immunohistochemical staining, interpreted to represent different subpopulations of cells at various phases of the cell cycle, were quantitated using PCNA immunohistochemistry. The proliferating index (PI), defined as the percentage of cells in the cell cycle (G1 + S + G2 + M), was more sensitive than the LI (S phase only) in detecting a chemically induced cell proliferative response.(ABSTRACT TRUNCATED AT 250 WORDS)
Related Articles
Misexpression of a bHLH Gene, cNSCL1, Results in Abnormal Brain Development
Developmental dynamics : an official publication of the American Association of Anatomists
NSCL1 is a basic helix-loop-helix transcription factor involved in the development of the nervous system. To elucidate its role in neurogenesis, we cloned chick NSCL1 (cNSCL1) and examined its expression pattern and the effect of its misexpression on brain development. cNSCL1 was predominantly expressed during active neurogenesis. Double-labeling experiments showed that proliferating neuroblasts in the ventricular zone lacked cNSCL1 expression and cells expressing cNSCL1 were located just outside the ventricular zone. Retroviral misexpression of cNSCL1 in chick embryos produced a brain with abnormal structure. While the forebrain of the embryonic day-12 (E12) brain appeared normal, the tectum was enlarged. The enlargement was likely due to an increase in cell proliferation, since more radioactivity was detected in this region of the brain after [3H]thymidine labeling at E9. The cerebellum, on the other hand, was reduced in size. Fewer cells were labeled with BrdU in the external granule layer (a secondary germinal layer required for cerebellum development) in experimental embryos than in the controls, suggesting that misexpression of cNSCL1 might interfere with cell proliferation in the external granular layer. Our data indicate that regulated expression of cNSCL1 is required for normal brain development. They also imply that cNSCL1 might be involved in preventing some postmitotic cells from reentering the cell cycle during neurogenesis.
bHLH; gene expression; neurogenesis; cell proliferation; differentiation; brain development
Related Articles
Sexual Phenotype Differences in zic2 mRNA Abundance in the Preoptic Area of a Protogynous Teleost, Thalassoma bifasciatum
McCaffrey, Katherine
Hawkins, Mary Beth
Godwin, John
PLoS ONE
The highly conserved members of the zic family of zinc-finger transcription factors are primarily known for their roles in embryonic signaling pathways and regulation of cellular proliferation and differentiation. This study describes sexual phenotype differences in abundances of zic2 mRNA in the preoptic area of the hypothalamus, a region strongly implicated in sexual behavior and function, in an adult teleost, Thalassoma bifasciatum. The bluehead wrasse (Thalassoma bifasciatum) is a valuable model for studying neuroendocrine processes because it displays two discrete male phenotypes, initial phase (IP) males and territorial, terminal phase (TP) males, and undergoes socially-controlled protogynous sex change. Previously generated microarray-based comparisons suggested that zic2 was upregulated in the brains of terminal phase males relative to initial phase males. To further explore this difference, we cloned a 727 bp sequence for neural zic2 from field-collected animals. Riboprobe-based in situ hybridization was employed to localize zic2 signal in adult bluehead brains and assess the relative abundance of brain zic2 mRNA across sexual phenotypes. We found zic2 mRNA expression was extremely abundant in the granular cells of the cerebellum and widespread in other brain regions including in the thalamus, hypothalamus, habenula, torus semicircularis, torus longitudinalis, medial longitudinal fascicle and telencephalic areas. Quantitative autoradiography and phosphorimaging showed zic2 mRNA hybridization signal in the preoptic area of the hypothalamus was significantly higher in terminal phase males relative to both initial phase males and females, and silver grain analysis confirmed this relationship between phenotypes. No significant difference in abundance was found in zic2 signal across phenotypes in the habenula, a brain region not implicated in the control of sexual behavior, or cerebellum.
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What's Happening at St. Paul's?
Refugee Family Update
Here is an update on our refugee family from Myanmar who resettled here on June 25. Mya Kyaw (pronounced mia chaw) and his family of 6 are doing great. They love the apartment and are very thankful for the furniture and all the things that we did for them. There are things that St. Paul's can continue to do to support this family. They are in need of some kids clothing and needs occasional transportation. Continue reading for more and to see what you can do….Continue reading
Adult Education (week of Jul 31)
This post gives information on upcoming adult education offerings. Continue reading below for details of Adult Education.Continue reading
What's Happening in Children's Ministries (week of Jul 31)
Weekly news from Christine Ingram, Director of Children's Ministries (firstname.lastname@example.com). Click Continue reading below.Continue reading
Arts and Crafts Festival Needs 2 Coordinators: Publicity and Facilities
The Arts & Crafts Festival for Outreach is Saturday, November 12, from 10:00 to 4:00. The festival committee needs two coordinators: one for publicity, and one for facilities coordinator. It's a fun event, for a very good cause. We have a great group, but we're looking for more wonderful volunteers. Please contact Sally Moller, firstname.lastname@example.com. Continue reading for job descriptions….Continue reading
Lobsters are Coming (Oct 1)
While you are enjoying summer's bounty of fresh tomatoes and corn, the warm sands of NC beaches, a little heat and humidity (well, maybe more than a little) and all the fun of summer, that's just a build-up to the "Rites of Fall:" football and LOBSTER FEST! We want to make sure you got the date on your calendar:
St. Paul's Annual LOBSTER FEST
Saturday, October 1
ASP Photos Posted
Movie Night (Aug 20)
Movie Night and Mission Trip Fundraiser August 20: Bring your lawn chairs and blankets and come watch a movie (title TBD) outside on a big screen. Think drive-in without a car! It's a family-friendly movie for a hot summer night. The screen will be set up in the courtyard. We will sell concessions (popcorn, candy, and drinks) and accept donations towards future mission or pilgrimage trips. The movie runs from 8:30-10:30 PM.
Consider Teaching Sunday School
Have you ever considered joining the Sunday School Teaching Ministry? If so, you are in luck as we have a few openings for Sunday School teachers for the 2016-2017 school year. Continue reading for more….Continue reading
Preschool Accepting Applications for 2016-2017
St. Paul's Preschool is currently accepting applications for the 2016-2017 school year. We have openings in most classes but the spots are filling quickly. Registration forms are available HERE. Please call 919-467-3788, or email the Preschool office, email@example.com with questions or to schedule a tour. Love, Laugh, and Learn with St. Paul's Preschool
St. Paul's is now on Instagram!
You can now find St. Paul's on Instagram (@stpaulscary)! Check out photos, videos, and stories from our vibrant community. Follow us and tag us in your photos.
The Diocese of North Carolina is also on Instagram (@episdionc), and we're using the hashtag #ThisIsDioNC to show prospective bishop candidates the faces, places, events and quiet moments that represent our diocese.

subset: commoncrawl


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Consulate General Of Argentina In Los Angeles: Crying
Producer: Laura Castro
Sound Design/Arrangement: Engineer: Jaime Zapatal
Advertising Agency: Conill Saatchi & Saatchi, Torrance, USA
Chief Creative Officer: Pablo Buffagni
Creative Director: Mario Granatur
Art Directors: Martin Donovan, Hernan Ibañez, Veronica Elizondo, Rob Casillas, Tony Rhi
Copywriters: Pablo Buffagni, Benjamin Levy, Abel Gonzalez, Jessica Cuevas
Agency Producer: Lourdes Caballero
Production Company: Parana Films Los Angeles, USA
Directors: Sebastian Leda, Francisco D'Amorim Lima
Producer: Raquel Ramirez / Tono Studios
Lighting: Daniel Karp
Post Production: Tunnel Post (Transfer)
Other Advertising Agencys: Fernando Brun/Martin Yarnezian/Luis Fernandez-Gil
22 comments
Finally, I saw this ad about a year ago or more on na Argentinean channel in the US. And, to be honest, I think I'll never forget this one!!!!!!!!!
Yeah advertising is dramatizing, this one does excellently on that ;p
Argentina vs Germany Quarter Final
What r the chances
Such a sad truth for Argentinian football....but great ad. I loved it - it brought smile to my face!
Let's just borrow some interest from soccer to make our film ad better!


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Mejora tu vida laboral (Book 2)Mejora tu relación con tus compañeros de trabajo y consigue un ambiente en la oficina más relajadoLidiar con personas conflictivas en la oficina requiere de paciencia, tolerancia y un plan estratégico que mejore tu estancia en tu trabajo. Para poder confrontar y desarmar a un colega tóxico, es preciso entender cuáles son los motivos que lo conducen a tener un comportamiento deleznable en el
Your Vibrant Business: Mind/Body/Spirit at WorkWhether you own your own business, have a 9-to-5 job, or are a stay-at-home mom, chances are that you're experiencing some overwhelm. Wouldn't you love to discover how to find calm and balance in all areas of your life using a mind/body/spirit approach?This book contains tips, tools, and techniques from 13 experts on subjects from finance to feng shui to help business owners enhance their mind
Your Neck - the 'Weakest Link'
Causes, Effects and Successful TherapyHow does your neck feel? Does it crackle and pop a lot? Do you often feel dizzy, or even feel you'll pass out when you turn your head sharply? Then perhaps you are also 'one of those cases' - a victim of 'the weakest link, your neck!' This is then something that you can't afford to ignore, for it could become a generator for the degradation of your health! It influences your body in various ways,
How Stress Hurts Us and How to Bounce BackComments like "I'm worried sick" convey the conventional wisdom that being "stressed out" will harm our health. Thousands of academic studies reveal that stressful life events (like a job loss), ongoing strains (like burdensome caregiving duties), and even daily hassles (like traffic jams on the commute to work) affect every aspect of our physical and emotional well-being. Cutting through a sea of
A Simple Guide to Office FitnessThis no-excuses fitness plan is the first to tackle the challenges of office workers who want to lose weight, get fit, improve posture, and combat stress--without ever leaving the comfort of their desk.People today are spending more time at work and less time being active, whether by sitting in front of a computer all day or in a car battling grueling traffic--or both! Working on the Ball solves
Effektives Zeitmanagement für mehr LebensqualitätWork-Life-Balance beschreibt kein Modephänomen, sondern eine fortwährende Herausforderung. Ein ausgeglichenes Verhältnis zwischen Arbeit und Freizeit ist sowohl für die Gesundheit als auch für die Zufriedenheit des Menschen wichtig. Yasmine Limberger erörtert im ersten Kapitel dieses shortcuts, wie man bei den zahlreichen E-Mails und anstehenden Meetings den Überblick behält und durch das Setzen
$4.99Web design is more than a job. It's a passion. However, keeping up with the immense pace at which the web industry is moving can feel quite intimidating at times and stress or even a burnout can strike all of us someday. With this eBook, we want to raise awareness for those aspects of the web industry which are not frequently talked about. It's not about frameworks, code or scripts for a change —
$3.99Can you have a life and be a doctor too?As a doctor you can have a fulfilled and balanced life both in and out of work.Would you like to have plenty of time for everything? Can you imagine what it would be like to be naturally tired after a satisfactory day's work, and have enough energy when you go home to spend quality time with your family and be able to look after your body, mind and spirit on
What You Can Do to Prevent Cancer: Practical Measures to Adjust Your Lifestyle and Protect Your HealthDid you know that one in three people in Ireland will develop cancer during their life?Or that an average of 30,000 new cases of cancer are diagnosed in Ireland each year? These are frightening statistics, but there are measures you can take to protect yourself and reduce your chances of developing the disease.In What You Can Do to Prevent Cancer, bestselling author John McKenna provides the
$15.19An estimated 7.8% of all Americans will experience post-traumatic stress disorder (PTSD) at some point in their lives. Roughly 5.2 million people have PTSD during the course of a given year. And PTSD can affect anyone - from war veterans and abuse victims to persons directly or indirectly traumatized by other catastrophes including crime, natural disasters, and serious accidents. Getting
What is Wellness? 101 Questions People Ask About Health and Wellness: and for which You've Gotta Have Answers!Wellness speaks of one's summary of life. In order for a person to say that he/she is living a full life, means he/she is able to enjoy it without stressors, illnesses and challenges.But in order to make sure that one lives this so-called full life, he/she must learn what wellness is really about, its dimensions and how it can be applied to everyday life. It is actually one thing to learn wellness
Und alles war ganz anders, als ich dachte
Chronik eines angekündigten Burn-outMachtspiele, Dominanz, Mobbing und Ausgrenzung treiben Gertrud Keller in die totale Erschöpfung. Im Sommer 2008 fühlt sie sich am Ende ihrer Kräfte. Körper, Geist und Seele sind ausgebrannt. Bewusst entscheidet sie sich gegen die Schulmedizin, gegen Medikamente und Klinikaufenthalte und sucht ihren persönlichen Weg aus dem Burn-out. Gertrud Keller erfährt dabei, dass sich Instinkte und


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2 months in Vietnam
2 months in Vietnam
If you had 2 months to explore, where would you head to? After Hanoi , I was thinking of heading inland from Hoi An to Kon Tum, Pleiku down to Dalat. Then hitting the coast at Nha Trang... or maybe staying inland ... before heading to HCMC, Mekong, Phu Quoc.
Also Sapa / North of Hanoi is an option (as well as the above), but it will be Dec time... and am concerned it will be too cold! ... any help greatly appreciated.
#1 chrispyrolls has been a member since 2/11/2006. Location: United Kingdom. Posts: 198
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2 months in Vietnam seems like overkill... Have you considered a side-trip to Cambodia or Laos? They're also warmer than northern Vietnam in the winter. I traveled Vietnam south to north in December/January and as soon as you were north of Nha Trang it was chilly... On the Halong boat trip we were freezing... don't count on doing any swimming or sea kayaking. Pack warm clothes and a coat with good wind protection if you're planning to do any motorcycle tours. It can be as low as 12C and the damp ocean air makes it even colder.
#2 idreamofdurian has been a member since 5/9/2008. Location: Singapore. Posts: 576
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was it british chilly... or singapore chilly?...:)
thanks for the response - this will be as well as Laos, Cambodia ... so a big trip
#3 chrispyrolls has been a member since 2/11/2006. Location: United Kingdom. Posts: 198
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I'm Canadian - believe me it was cold!
There's endless things to do in Vietnam... Mui Ne was a surprising highlight of my trip (enjoyed it much more than Nha Trang). Other than the beach, it's a really nice area to rent a bike and explore (not much traffic) and you can go "dune-surfing" which is a pretty unique experience.
#4 idreamofdurian has been a member since 5/9/2008. Location: Singapore. Posts: 576
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blimey it must be cold then! ... thanks very much
#5 chrispyrolls has been a member since 2/11/2006. Location: United Kingdom. Posts: 198
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2 months in Vietnam sounds so wonderful. Those that think the nation can be 'seen' in a few weeks leave me gobsmacked.
But, then, so many people just spend their time in buses/trains/planes/taxis, etc. travelling between cities, and when in cities, just seek out nightclubs.
I could spend 2 months just south of the Mekong!
You haven't indicated your focus for the Vietnam trip. Maybe if you indicated what you want to focus upon, others could better assist.
#6 brucemoon has been a member since 27/12/2008. Location: Australia. Posts: 1,941
'2 months in Vietnam seems like overkill'
jep. I live here since June 2007 and I hate Vietnam.
My insider recommendation: Phu Quock!!! It is a small island with a tiny airport, is is ubelivaby beautiful, clear water. There are only less flights to there. I've been there, i know a german family living there they have a resort with like 5 huts, (2 beds per hut), there are less tourists there when you go before June.
#7 Phen0m has been a member since 2/5/2009. Posts: 6
Sorry i spelled Phu Quock wrog: it is Phu Quoc
#8 Phen0m has been a member since 2/5/2009. Posts: 6
I would just buy or rent a motorbike , and see it for yourself , after 2 months you should be able to visit every province. I like Quang Nam Near Tam ky good room prices cheaper food and nice nearby beach, no tourists , almost never.
#9 travworld has been a member since 9/7/2007. Location: United States. Posts: 23
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June has traditionally been a slow month for teaching as are the rest of the summer months. We will have gone 5 weeks between MAD Live Events. I was blessed to preach at the Everett Hills Baptist Church in Maryville, TN this month. Later in the month I did my only MAD Live Event in June. We were privileged to partner with "106.7 The Light", Billy Graham's other radio station in Knoxville, TN to do the event. 105 people showed up and we praise God that 3 more people gave their lives to Jesus Christ!
After the MAD Live Event, we packed up and headed west to Colorado and stopped to see my brother Doug and his wife Diane. They just moved into their new home in Breckenridge. What a beautiful house and view of the Rockies! From there we left and completed the journey to Utah to host this year's Ultimate Adventure trip. We have participants from PA, NJ, and NY, 10 in all. More info on the trip will be on our Facebook page and in the July report.
I did have a number of opportunities to share the Gospel with individuals this month. One of my favorite ones was when we had some landscapers come to remove some intrusive bushes that were growing behind and into our electric service and siding. They did great work and I had a wonderful opportunity to share the Gospel with them. I used the Way of the Master to show them their sin and need for a Savior and gave them Gospels of John to read. They were very attentive and asked a number of questions. Based on the expressions on their faces and questions they asked I am certain the Holy Spirit was at work in their hearts. Marty is a US Army veteran and a Native American. Chris is from the Bayou. I gave them my card and asked them to call me with questions. Chris told me he would read the entire Gospel tonight when he went home. Please pray for them as they come to mind.
"Hey Don and Cathy! Today was a great time to read your ministry report as I have the opportunity in a couple of hours to share the gospel with the man who is coming to fix my car in a couple hours. Your prayer request that God would continue to help you recognize and take advantage of the divine appointments really applies to me recently. I´m waking up from another stagnant season, and thrilled to see God answering my prayer for a witnessing buddy. Being on Nantucket (I came to listen to Don in Brewster, MA last Summer) is a totally different and intimidating culture for me. What a spiritual battle! Praying for Matt who was mentioned in the testimony and praying for your leg, Don. Can´t wait to meet you someday Cathy! Keep up the good work friends!"
• July 1-7 - Ultimate Adventure trip to Utah
• July 16 - MAD Live Event - Mountain View Community Church, Culpeper, VA
• July 20-23 - Present 3 mini MAD seminars and have a booth display - Kingdom Bound Christian Music Festival, Darien Lake, NY
• July 28 – Arthroscopic surgery to repair the completely torn Meniscus in my left knee
• July 30 - MAD Live Event with Pillar College, Somerset, NJ
• July 31 - MAD Live Event - Crossroads Community Church, Indian Mills, NJ
• August 6 - Walk thru the Bible Old Testament seminar - Cape Community Church, Cape May Courthouse, NJ
• August 6 - Preach at Saturday evening worship service - The Lighthouse Church of the Christian and Missionary Alliance, Cape May Courthouse, NJ
• August 7 - Preach at both Sunday morning worship services - The Lighthouse Church of the Christian and Missionary Alliance, Cape May Courthouse, NJ
• August 20 - MAD Live Event - Crestview Baptist Church, Canton, NC
• August 28 - Preach 3 times at Sunday morning worship services - Valley Forge Baptist Temple, Valley Forge, PA
• Sept 6 - Walk thru the Bible Old Testament Seminar - Cape Community Church, Cape May Courthouse, NJ
• Sept 6 - Preach at the Saturday Evening Service - Lighthouse Church of the C&MA, Cape May Courthouse, NJ
• Sept 7 - Preach at both Morning Services - Lighthouse Church of the C&MA, Cape May Courthouse, NJ
• Sept 20 - MAD Live Event - Crestview Baptist Church, Canton, NC
• Sept 28 - Preach at both Morning Services and the Evening Service at Valley Forge Baptist Temple, Valley Forge, PA
• For safe travels for Cathy, me, my granddaughter Maya, and those attending the 2016 Ultimate Adventure trip to Utah. Due to the impending surgery on my knee, I will not be able to lead most of the trip. More importantly, we have a family of 4 who are coming who don't know the Lord. Please pray that the truth of God's word will take root in their hearts and salvations will occur.
• We are really in need of your prayers AND help filling the schedule for the fall. It amazes me that it progressively gets harder and harder to get commitments, and even return calls from pastors. I struggle with frustration over the lack of interest and response.
• For us to be faithful with the Divine appointments God brings us each day
• I would like to start a Bible study in our neighborhood. Please pray that we will find receptive hearts willing to come and learn.
Love in Christ,
Cathy and Don


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Please enter a valid mileage
Curious about the newer model? Check out the 2017 Mercedes-Benz C-Class
By KBB.com Editors
The C-Class offers young drivers a way to join the exclusive Mercedes-Benz family without spending an arm and a leg. Although priced slightly higher than similarly equipped models from Audi, Infiniti, Saab, Cadillac and Lexus, the C-Class can certainly match these vehicles in the areas of luxury, performance and value. The availability of a slick-shifting six-speed manual with the C230 and C350 Sport trims is a must for the driving enthusiast and is offered as a no-charge alternative to the seven-speed automatic. Those not concerned by such matters will find much to like in the C-Class, including a number of desirable features such as the 4MATIC all-wheel drive and a new Flex-Fuel V6 capable of running on E85 or gasoline.
If you're looking for the quality and prestige that comes from the Mercedes-Benz brand at a price comparable to some top-of-the-line family sedans, the C230, C280 and C350 are the way to get it.
Prestige has its price. The C-Class is tight on rear-seat legroom and can cost thousands more than comparably equipped European, American and Japanese rivals.
The C230 receives a new E85-compliant engine, while the C280 and C350 Luxury models gain power front seats and a power-adjustable steering column with memory settings for up to three drivers. C230 and C350 Sport models receive new 17-inch wheels, an AMG rear deck spoiler and dual AMG exhaust pipes.
The top-of-the-line C350 definitely feels tighter and more in touch with the road than most of its luxury counterparts. Yes, the ride is a bit firm and can even be...
... rough over broken pavement and untended expansion joints, but it's a small price to pay for the benefits a taut suspension yields.
The C350's six-speed manual is a vast improvement over previous Mercedes-Benz manuals, with much tighter gates facilitating shorter shifts. The 3.5-liter V6 engine is an absolute dream. It responds willingly to the slightest throttle input, calling upon its ample low-end torque to shoot you across busy intersections or to overtake slower-moving traffic.
Panoramic Glass Sunroof
The C-Class's panoramic glass sunroof covers the entire interior and includes an electric sunshade for both the front and rear panels.
Quality Interior
The C230, C280 and C350 models offer a Mercedes-quality interior at a surprisingly affordable price.
Inside you'll find a comfortable set of sport bucket seats complete with fore and aft adjustable headrest and controls for the seat-bottom angle and seat height adjustment. From the driver's seat, you'll find a logically appointed instrument panel, devoid of overly complicated touch-screen menus and confusing switchgear. The instrument cluster features a digital trip computer neatly tucked between the speedometer and tachometer. Steering wheel-mounted controls regulate the audio and information screens.
The C-Class is a Mercedes-Benz product through and through. With overtones of E- and S-Class clearly visible in the design, the C-Class fits right into the Mercedes family and should more than satisfy any entry-level buyer. The Sport models offer a more distinct look than their Luxury counterparts, with sporty six-spoke alloy wheels and numerous details borrowed from Mercedes-Benz's AMG performance division.
The C230 Sport features a six-speed manual transmission, dual-zone automatic air conditioning, four-wheel anti-lock disc brakes (ABS), front and rear fog lights, dual heated power mirrors with turn signals, AM/FM/CD stereo, front and rear side-impact and head-curtain airbags, stability control, cruise control, sport suspension, 17-inch alloy wheels and heated windshield wipers. The C280 and C350 Luxury models add more powerful V6 engines, 16-inch wheels and more standard equipment plus the choice of rear-wheel or all-wheel drive.
Popular C-Class options, which are typically grouped into packages, include a panoramic glass sunroof, rain-sensing wipers, in-dash six-disc CD changer, audio upgrades, COMAND navigation system, heated front seats and a power rear sunshade.
The C230's new 2.5-liter powerplant is a definite improvement over the old supercharged four-cylinder and its Flex-Fuel versatility makes it ideal for areas that supply E85. The more powerful V6s found in the C280 and C350 suit this car's image, but buyers will pay a price, first at the dealership and then at the gas pump.
2.5-liter V6
201 horsepower @ 6200 rpm
181 lb.-ft. of torque @ 2700-5000 rpm
EPA city/highway fuel economy: 14/18 (E85), 19/25 (Gasoline)
3.0-liter V6
228 horsepower @ 6000 rpm
221 lb.-ft. of torque @ 2700-5000 rpm
EPA city/highway fuel economy: 22/28 (RWD), 19/26 (AWD),
3.5-liter V6
268 horsepower @ 6000 rpm
258 lb.-ft. of torque @ 2400-5000 rpm
EPA city/highway fuel economy: 20/28 (RWD manual), 20/29 (RWD automatic), 19/24 (AWD automatic)
To compare current market prices, check out KBB.com's car classifieds to view the new and used Mercedes-Benz C-Class vehicles for sale near you.
Based on 831 Ratings for the 2001 - 2007 models.Review this car
By Mike on Wednesday, June 8, 2016
I own this car - My approximate mileage is 127,000overall rating 9 of 10rating details
Pros: "It handles like a dream sporty or luxuries ride"
Cons: "None"
Likely to recommend this car? (1-10): 10
"I have meant to buy a new car but I love this one and its performance.It rides like new and have had excellent care. It has never let me down and if you will give it a little love it will give you back ten fold."
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By Tez on Wednesday, December 30, 2015
I own this car - My approximate mileage is 190,000overall rating 7 of 10rating details
Pros: "Great Price, fun to drive, good first time car."
Cons: "Have to maintain car, and can be costly!"
Likely to recommend this car? (1-10): 8
"The condition of my 2002 Mercedes Benz C240 is considerably good. It has some rust spots on the bottom of the driver and passenger doors. The driver side back window motor does not work, so motor needs to be replaced. The window won't let down because of it. The tape player inside the vehicle is very old, but does function. Now thats the bad stuff! On the bright side the vehicle has: power: locks, seats, windows, air condition, heat, sunroof, leather interior, radio works, and it drives very good. There are not leaks under the hood, and I have kept the vehicle maintenance up since i owned the vehicle. This would be a very good first time vehicle or just extra vehicle to have around. It is so fun to drive in the Spring and Summer time!"
9 people out of 12 found this review helpful
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By Iris on Monday, October 26, 2015
I own this car - My approximate mileage is 60,000overall rating 10 of 10rating details
Likely to recommend this car? (1-10): 10
"I'm the only owner of car,I bought it new. Always indoor parking, clean, no rust, never repair, no accidents, only new battery and 4 new tires. I'm a senior widow and only used car for church and food shopping, only 60,000 miles."
24 people out of 31 found this review helpful
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By brokecollegestudent on Saturday, September 19, 2015
I own this car - My approximate mileage is 101,900overall rating 10 of 10rating details
Pros: "fun to drive, relaxing to use, runs very quiet"
Cons: "possible expensive fixes"
Likely to recommend this car? (1-10): 9
"Ok, so I work at a factory. Bust my butt for 8-12 hours. My back hurts from being on my feet all day, but then I step into my 06 c280. Instant relief and support on my back when I sit down, and the seat is insanely comfortable. The ride is so smooth, I can't even tell when I hit bumps or run over rocks - and there's a lot of those around where I live. I have gotten many compliments from friends and jealous looks from my family, always a plus because I hate half of them. The maintenance, when it needs to be done, can be quite pricy. It's worth it to look around where you live for a decent mechanic that does good work over going to the Mercedes dealership and have to spend $800 on an oil change. I had to replace the rear diff. oil because it was running low, but I went to Napa and got the kind I needed from an off brand for $20. That was a quick fix. I also visit a fast lube and they do a lot of small adjustments and quick fixes for no charge, which helps a lot. They like it when I visit because they like checking it. (I live in a small town you see, you don't see a lot of cars like mine around a place like this.) Aside from basic maintenance and some tiny little issues that were fixed in seconds or minutes, this car has been a dream to own. I'm not sure how long I'm going to keep it, but the insurance cost is a lot lower than I expected it to be, and I got it for about 10% what it was worth when it was brand new. Definitely worth the seemingly hefty price tag, because you're getting a well-built, sturdy car with decent mileage. Be sure you get as many service records as possible. Luckily for me, the previous owner kept a record of all the service he had on it and it runs perfectly. I think the only thing I would change about it is the radio, but that's because it's 2015 and I want to have GPS navigation and stream internet radio. Guess it's off to the car accessory place! (But seriously, the radio works just fine, and the sound quality is amazing and sounds just like a pair of brand new speakers.) Overall, easily the fanciest car I have ever owned, and is friendly on a broke college student's budget. Definitely worth it if you're okay with the cost of maintaining it. Remember though - it's not very often that you need to have it done."
24 people out of 31 found this review helpful
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By wogga on Tuesday, September 15, 2015
I own this car - My approximate mileage is 90,000overall rating 9 of 10rating details
Pros: "inexpensive MB-class entry point"
Cons: "what you save in purchase, you pay in maintenance"
Likely to recommend this car? (1-10): 9
"I'd buy this car again, though maintenance (at a specialist independent mechanic) and the few things that break will probably set you back $1500-$3000 a year, depending on labor rate and luck. It drives quite well (if all your suspension and handling gear are fixed right), but it is not a BMW. It is just posh enough to feel satisfactorily different than our family's Japanese, US and even VW cars. Ours has been very reliable, even though it was a rebuilt title car when I got it. Don't buy a rebuilt car--ever; I just got lucky. Because it's rebuilt though, resale value is poor; that means you'd better plan to own it forever to get the value of all the pricey parts you'll be putting in over the years. One front axle cost me $1800 for the part alone - close to $2,500 installed I think. My wife drives it mostly, loves it and notices she's treated better by other drivers compared to when she drives our Japanese or US cars. The MB sign sticking up on the hood looks very nice. Irritants: Beware an inexpensive used MB! Keys are very expensive if you need new ones, but they are very pretty. You really should use a MB specialist with this car; MB have made it difficult for your average mechanic (or you) to do much on it well. The car is full of nice things that I won't even bother mentioning. EG: in 'C' transmission mode, you will not roll backwards when stopped pointing up hill. The mirrors don't fold in like every other decent european car--I was told by an MB official 'that's because you bought (only) a C-class car'....%$#^#!!!! The ABS when it does come on (on ice, snow etc) in my car is quite a nasty sound/feel, like clicking, grating things... The rear wiper assembly on the tailgate window isn't good at it's intended job Rear seat leg room is worse than my mom's Subaru Imprezza, though quieter and nicer. Rain sensing wiper mode was timed by someone who can see through raindrops better than I can! Oh for manually selectable 'intermittent wipers'. Oh and slow mode is to slow and fast mode is 'holy moley it's monsoon season' fast. You cannot tow anything. You cannot mount a rear bike rack. A car with a 3.2 liter should be able to tow 2000 or more. A wagon should be designed to be able to take a wagon-mounted bike rack."
8 people out of 15 found this review helpful
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By Veronica on Monday, September 14, 2015
I own this car - My approximate mileage is 107,000overall rating 9 of 10rating details
Pros: "Sportiness, most comfortable seats, dependable."
Cons: "costs a bit more in maintenance. Worth it."
Likely to recommend this car? (1-10): 2
"I still own this car. I drive a lot from N.Y to Canada and this car has been a dream in comfort, peppiness and dependability. I hate to see it get old. I would buy it all over again if there was an option. Unfortunately, I am not thrilled with the new Mercedes overcomplicated ugly touch screen design. Especially the gear shift location being in the steering wheel now."
10 people out of 18 found this review helpful
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HTML5 Projects: Engaging Ecommerce
The HTML5 Projects series combines HTML5, CSS3, and recent JavaScript API technologies to work—enhancing your web projects with interactivity and multimedia. This installment shows you how to build features that allow visitors to your online store to filter their selections interactively via sliders, drag items onto wish lists, and automatically save them locally. Author Joseph Lowery uses this scenario as an opportunity to introduce concepts such as HTML5 form elements with JavaScript enhancements, local content storage, and drag-and-drop.
- Including range form elements
- Modifying the display interactively
- Establishing JavaScript functions
- Dragging items to wish lists
- Storing wish list items locally
- Saving and retrieving list items
- Subject:
- Web
- Software:
- HTML JavaScript CSS
- Author:
- Joseph Lowery
- Hi, I'm Joe Lowery, and I'm so happy you've joined me for HTML5 Projects Engaging Ecommerce. First, I'll show you how to use the coolest of the many new form elements in HTML5, the range input type, to filter catalog items interactively. Next, we'll bring drag and drop into play, so site visitors can pick their favorite products quickly and with a little flair. Finally, I'll demonstrate how to automatically store selected favorites for the user, via the cutting edge Java Script method, known as local storage.
As you go through the course, keep in mind that the ecommerce catalog is just one example of how HTML5 can greatly enhance your users' experience. Alright, let's get started.
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NASA to Launch Ocean Wind Monitor to ISS
Pasadena CA (JPL) Feb 01, 2013
In a clever reuse of hardware originally built to test parts of NASA's QuikScat satellite, the agency will launch the ISS-RapidScat instrument to the International Space Station in 2014 to measure ocean surface wind speed and direction. The ISS-RapidScat instrument will help improve weather forecasts, including hurricane monitoring, and understanding of how ocean-atmosphere interactions influence Earth's climate. "The ability for NASA to quickly reuse this hardware and launch it to the space ... read more
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Check prices on hotels in Newport
Most Popular Hotels in Newport
Get 200 Nectar points when you book your hotel
Simply enter your Nectar card number when prompted. Your points will be credited 30 days after you return from your trip.
Lilleshall National Sports & Conferencing Centre
Near Newport, Newport TF10 9AT4.0 out of 5.0
Located in Lilleshall, this bed & breakfast is within 9 mi (15 km) of Weston Park, The International Centre, and Telford Ice …
Furnace House
64 Furnace Lane, Telford TF2 7JE3.0 out of 5.0
Situated in Donnington, this bed & breakfast is 3 mi (4.9 km) from New Bucks Head Stadium and within 6 mi (10 km) of Telford …
Park Inn by Radisson Telford
Forgegate, Telford TF3 4NA4.0 out of 5.0
Situated in Telford, this eco-friendly hotel is within a 15-minute walk of Telford Shopping Centre and The International …
Offley Grove Farm
Adbaston, Stafford ST20 0QB3.0 out of 5.0
Situated in a rural location, this bed & breakfast is within 16 mi (25 km) of Weston Park, New Bucks Head Stadium, and The …
Days Inn Telford
M54 Junction/4, Shifnal TF11 8TG2.0 out of 5.0
Situated in Shifnal, this hotel is within 3 mi (5 km) of Telford Shopping Centre, The International Centre, and Telford Ice …
Telford Whitehouse Hotel
Watling Street, Telford TF1 2NJ4.0 out of 5.0
Located in Wellington, this spa hotel is 0.1 mi (0.1 km) from New Bucks Head Stadium and within 6 mi (10 km) of Telford …
Ramada Hotel Telford Ironbridge
Forgegate, Telford TF 3 4NA3.0 out of 5.0
Located in the heart of Telford, this hotel is within a 15-minute walk of Telford Shopping Centre and The International …
Holiday Inn Telford - Ironbridge
Telford International Centre, Telford TF3 4EH3.0 out of 5.0
This family-friendly Telford hotel is located in the business district, within a 15-minute walk of Telford Town Park …
Mercure Telford Madeley Court Hotel
Castlefields Way, Telford TF7 5DW3.0 out of 5.0
Situated on a lake, this hotel is within 2 mi (3 km) of Blists Hill, Iron Bridge, and Ironbridge Museum. Coalport China …
The Shakespeare Inn
High Street, Telford TF8 7HT3.0 out of 5.0
Situated in Jackfield, this historic inn is within a 15-minute walk of Coalport China Museum and Maws Craft Centre. Blists …
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'BULB' PhotoAccess Members | 'A Closer Look' Personal Photography Project exhibition | 21 July - 7 August 2016 | Exhibitions
(Image: Lorna Sim, Alison (dancer/choreographer Alison Plevey) (detail), 2016, archival inkjet on photo rag, 10 x 8 inc)
PhotoAccess presents in the Huw Davies Gallery:
BULB, a PhotoAccess members' exhibition, and A Closer Look by PhotoAccess' Personal Photography Project participants.
Opening on Thursday 21 July 2016 at 6pm
Exhibitions to be opened by Sean Davey, Director of The Photography Room, photographer and PhotoAccess Tutor.
Exhibition dates: 21 July - 7 August 2016
BULB: photographs of movement
BULB explores movement: movement in pictures, movement in life. A photograph freezes the world around us, but it does not mean that the world within the photograph is frozen. When set to 'bulb' mode, a camera's shutter will remain open for as long as the photographer's finger remains on the release button to create images full of motion and blur. BLUB showcases work by 30 PhotoAccess members: Stephen Best, Ulli Brunnschweiler, Joseph Cali, Alan Charlton, Tom Chesson, Lori Cicchini, Casey Crockford, Tod Davis, Marwan El Hassan, Jennifer Everart, Ngaio Fitzpatrick, Tony Fleming, Susan Henderson, Gilbert Herrada, Margaret Kalms, Angus Kendon, Paul Livingston, Kate Luke, Thea McGrath, Nadia Mcleish, Don McLeod, Scot Newman, Judy Parker, Brian Rope, Lorna Sim, Kim Sinclair, Neil Stafford, Brian Stewart, Lachlan Thompson and Mervyn Wright. Catalogue
A Closer Look
In 2015/16 PhotoAccess ran the eleven-month long Personal Photography Project workshop (culminating in this group exhibition A Closer Look) which provided a supportive environment for Canberra-based emerging photographers to develop a project of their choice. A Closer Look showcases works by participants of the Personal Photography Project: Fiona Bowring-Greer, Susan Clarke, Karen Coombes, Lyndal Curtis, Zelda Green, Rob Lee, Bronwyn McNally, Kleber Osorio, Ian Skinner, Gordon Taylor and Michael Ye. Catalogue
(Image: Zelda Green, Ade, 2015, inkjet print. 25.0 x 20.0 cm)
Winter 2016 Education Program
Classes in analogue and digital photography, studio lighting, cinematography and 19th Century photographic processes, for all experience levels are now open for enrolment. Gain new skills, expand your knowledge, or learn new techniques at PhotoAccess this Winter! Click here to find out more.