Gold Labeling Research Articles

Characterization of avian thymic hormone and chicken parvalbumin 3 target cells

Avian thymic hormone (ATH) is a β-parvalbumin produced by epithelial cells in the thymic cortex and in the eyes of chickens. Chicken parvalbumin 3 (CPV3) is a homologous protein produced in the thymus and in hair cells of the chicken ear. ATH circulates in the blood on a five-day cycle and stimulates cell-mediated immunity when administered to young chickens. We report the identification of target cells for ATH and CPV3 and the immunophenotype of target cells for ATH. Newly hatched chicks were injected intracoelomically with ATH and killed 5, 10, 15 or 20min later. Naïve chickens also were killed at 1, 7 and 14days of age. Various tissues were examined by EM for the presence of either ATH or CPV3 using colloidal gold labeling. Gold particles were initially present on plasma membranes of lymphocytes in T cell areas of spleen and cecal tonsils from the chicks injected with ATH, internalized within 10min, and accumulated in nuclei by 20min. Immunofluorescence staining also identified the presence of ATH in T cell areas of spleen and cecal tonsils. Target cells labeled for ATH were immunophenotyped by double labeling. They were positive for CD3, CD8 and the lymphocyte receptor TCR-1, a phenotype characteristic of cytotoxic γδ T cells. Some of the target cells in the spleen were TCR-3 positive. Targeting of lymphocytes by CPV3 indicated that it may also be an immunomodulating hormone.

Biaxial Flexural Strength of High-Viscosity Glass-Ionomer Cements Heat-Cured with an LED Lamp during Setting

Adding heat to glass ionomers during setting might improve mechanical properties. The aim was to compare the biaxial flexural strength (BFS) between and within four glass ionomers, by time of exposure to a high-intensity LED light-curing unit. Materials and methods. Samples of Fuji 9 Gold Label, Ketac Molar Easymix, ChemFil Rock, and the EQUIA system were divided into three treatment groups (n = 30): without heating (Group 1), heated with LED lamp of 1400 mW/cm2 for 30 s while setting (Group 2), and heated with LED lamp of 1400 mW/cm2 for 60 s while setting (Group 3). Samples were stored for 48 hours in distilled water at 37°C until tested. BFS was tested, using a universal testing machine at a crosshead speed of 1 mm/min. Data were analyzed, using ANOVA test with the Bonferroni correction (α = 0.05). Heating the glass-ionomer cements with an LED curing light of 1400 mW/cm2 during setting for 30 s increased the BFS value of all GICs. No statistically significant difference in mean BFS scores was found between the EQUIA system and ChemFil Rock at 30 s and 60 s. The mean BFS value was statistically significantly higher for the EQUIA system and ChemFil Rock than for Fuji 9 Gold Label and Ketac Molar Easymix at all exposure times.

Evaluation of the multimetal deposition process for fingermark enhancement in simulated operational environments

Multimetal deposition is a fingermark development process that has been adapted from colloidal gold labelling used for biological applications. It has been identified as having particular potential for the development of fingermarks on cling film wrapping materials, where currently used processes have very low success rates. This paper focuses on exploring whether multimetal deposition continues to be effective when the cling film surface is exposed to simulated operational conditions including exposure to drugs contamination, immersion in water and physical distortion. The results indicate that multimetal deposition continues to develop fingermarks after such exposure and there are potential benefits in implementing the process more widely.

Fluoride Release from Glass Ionomer Cements: Effect of Temperature, Time Interval and Storage Condition

ABSTRACTObjectivesFluoride plays a pivotal role in oral health promotion and is the corner stone in the prevention of dental caries. The purpose of this study was to evaluate the effects of temperature, time interval and storage conditions on fluoride release by three commercial glass ionomer cements (GIC).Materials and MethodsThree commercial glass ionomers: GC Gold Label Universal Restorative (conventional), GC Gold Label Light-Cured Universal Restorative and GC Fuji VII (GC Corporation, Tokyo, Japan) were used to investigate fluoride release. The 6 × 2 mm disk-shaped specimens prepared from each material were immersed in distilled water or artificial saliva at temperatures 4°C, 37°C or 55°C. The amount of fluoride release from these specimens was studied for the period of 1, 7, 14 and 28 days with the help of fluoride selective ion electrode.ResultsThe amount of fluoride release was significantly (p < 0.001) high in distilled water when compared to artificial saliva in all the three types of GIC studied. The highest level of fluoride release was observed on the first day of the study, followed by days 7 and 14, with least release on days 28. It was also observed that at 55°C, the amount of fluoride release was significantly (p < 0.001) high in all three GIC. However, there was no significant difference among the three GIC studied.ConclusionThe amount of fluoride release depends on temperature, time interval and storage condition. The result from the study concludes that GIC in the oral cavity serve as fluoride reservoir and contribute a low fluoride release in oral fluids.Clinical significanceThe clinical use of GIC that release fluoride is relevant mainly in patients at risk of or with caries activity, thereby preventing initiation of secondary caries and failure of restorations. It may also be important in developing regimes for improving the delivery of tropical fluoride products. This study would be helpful to make some considerations about clinical indication and longevity of restorative materials studied.How to cite this articleMadhyastha P, Kotian R, Pai V, Khader AMA. Fluoride Release from Glass Ionomer Cements: Effect of Temperature, Time Interval and Storage Condition. J Contemp Dent 2013;3(2):68-73.

Abstract P6-06-03: Caveolin-1: A potential mediator of RhoC GTPase driven Inflammatory breast cancer cell invasion.

Abstract The proposed study focuses on Inflammatory Breast Cancer (IBC), which is one of the most aggressive forms of locally advanced breast cancer. IBC is an understudied disease in terms of identifying various molecular entities that could potentially be responsible for the aggressiveness of this disease. Recent advancements reveal that IBC has a unique molecular profile when compared with non-IBC. Caveolin-1 is found to have opposite expression pattern in IBC versus non-IBC. Our lab has shown that Caveolin-1 is found to be over-expressed in IBC whereas in non-IBC its expression is much reduced compared to normal mammary epithelial cells. Caveolin-1 is known to be associated with specialized lipid rafts in the plasma membrane called ‘caveolae’. ‘Caveolin scaffolding domain (CSD)’ from amino acids 82–101 concentrates variety of signaling molecules such as growth factor receptors, protein kinases, heterotrimeric G proteins and Rho GTPases. Thus it is known as a potential regulator of many signaling pathways. Rho GTPases are members of the Ras superfamily of small GTP binding proteins involved in cytoskeletal rearrangements during cell motility and invasion. Our lab has previously shown that RhoC GTPase is over expressed in IBC and that exogenous expression of RhoC GTPase produces motile and invasive phenotype in human mammary epithelial cells (HMECs) similar to SUM149 IBC cells. Thus the overall objective of this study is to determine if caveolin-1 over expression mediates the RhoC GTPase driven invasive phenotype of IBC via its scaffolding action. The first objective of this study is to elucidate the effect of caveolin-1 over expression on SUM149 IBC cell invasion. This was done by down regulating caveolin-1 to a level that is comparable to human mammary epithelial cells (HMECs) by using si caveolin-1. With the same si caveolin-1 or exogenous CSD synthetic peptide, we performed Matrigel invasion assays followed by cell viability and proliferation assay. Si caveolin-1 did not produce significant changes in cell viability over the period of four days as well as in Ki-67 staining which was used as a proliferation marker. Caveolin-1 down regulation reduced cell invasion by 90% compared to untreated cells. Similarly, exogenous CSD synthetic peptide treatment reduced SUM149 cell invasion significantly. In order to understand caveolin-1 localization in SUM149 cells, we performed immuno gold labeling of caveolin-1 by transmission electron microscopy as well as sucrose gradient centrifugation. Both these methods showed distinct pool of cytoplasmic caveolin-1 in addition to plasma membrane associated caveolin-1. Co-localization of caveolin-1 and RhoC GTPase was studied by co-transfecting cells with GFP-caveolin-1 and RFP-RhoC GTPase. Thus, this data suggests potential role of caveolin-1 over expression in regulating IBC cell invasion. Due to its ability to concentrate variety of signaling molecules, caveolin-1 could act as a principle regulator of several downstream events that make cells motile and invasive through RhoC GTPase activation. Thus studying caveolin-1 localization and effect of its regulation on the signaling pathways could help understand the important molecular mechanisms in IBC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-06-03.

Importance of membrane selection in the development of immunochromatographic assays for low-molecular weight compounds

This study was performed to demonstrate the importance of selecting an appropriate membrane when developing immunochromatographic assays (ICAs) for the sensitive detection of low-molecular weight compounds. Based on our findings, we propose a theoretical basis for selecting such a membrane. When eluting the sample solution for the competitive ICA using colloidal gold label for low-molecular analytes, the degree of binding inhibition is proportional to the collision frequency between the antibody-colloidal gold (Ab–CG) and analyte before Ab–CG binding to the capture antigen and a higher concentration of pesticides around the Ab–CG leads to a greater degree of inhibition. Therefore, we propose that the relative migration speed of the analyte and Ab–CG on the test strip is critically important for selecting a membrane in the development of sensitive competitive ICAs. We developed a novel method to estimate such a relative migration speed. We demonstrated the applicability of this proposal by using it to select an appropriate membrane for the development of an ICA of the pesticide diazinon.

Endocytosis in Trypanosoma cruzi (Euglenozoa: Kinetoplastea) epimastigotes: Visualization of ingested transferrin–gold nanoparticle complexes by confocal laser microscopy

Here we describe the visualization by confocal microscopy of ingested gold (15nm)-labeled transferrin in epimastigote forms of the protozoan Trypanosoma cruzi. Intracellular gold labeling was evident at two sites, which represent the bottom of the cytopharynx and the reservosomes. The gold tracer was best observed by confocal microscopy by using the 633nm excitation wavelength. Intracellular gold clusters larger than 60nm could be visualized by either gold reflection (light scattering) or photoluminescence modes. The gold reflection mode, the gold photoluminescence mode and the anti-transferrin immunofluorescence image of gold-labeled transferrin showed co-localization, thus demonstrating that the gold visualization modes did not represent artifacts or mislocalization of the biomarker. Visualization of protein–gold nanoparticle complexes by confocal microscopy thus emerges as a promising imaging tool to explore the endocytic pathway in trypanosomes and other cell types, as well as to perform immunolocalization studies using gold-labeled secondary antibodies.

Trypanosoma brucei 20 S Editosomes Have One RNA Substrate-binding Site and Execute RNA Unwinding Activity

Editing of mitochondrial pre-mRNAs in African trypanosomes generates full-length transcripts by the site-specific insertion and deletion of uridylate nucleotides. The reaction is catalyzed by a 0.8 MDa multienzyme complex, the editosome. Although the binding of substrate pre-edited mRNAs and cognate guide RNAs (gRNAs) represents the first step in the reaction cycle, the biochemical and biophysical details of the editosome/RNA interaction are not understood. Here we show that editosomes bind full-length substrate mRNAs with nanomolar affinity in a nonselective fashion. The complexes do not discriminate-neither kinetically nor thermodynamically-between different mitochondrial pre-mRNAs or between edited and unedited versions of the same transcript. They also bind gRNAs and gRNA/pre-mRNA hybrid RNAs with similar affinities and association rate constants. Gold labeling of editosome-bound RNA in combination with transmission electron microscopy identified a single RNA-binding site per editosome. However, atomic force microscopy of individual pre-mRNA-editosome complexes revealed that multiple editosomes can interact with one pre-mRNA. Lastly, we demonstrate a so far unknown activity of the editing machinery: editosome-bound RNA becomes unfolded by a chaperone-type RNA unwinding activity.

Mechanism of Evenness Interrupted (Evi)-Exosome Release at Synaptic Boutons

Wnt signaling plays critical roles during synaptic development and plasticity. However, the mechanisms by which Wnts are released and travel to target cells are unresolved. During synaptic development, the secretion of Drosophila Wnt1, Wingless, requires the function of Evenness Interrupted (Evi)/Wls, a Wingless-binding protein that is secreted along with Wingless at the neuromuscular junction. Given that Evi is a transmembrane protein, these studies suggested the presence of a novel vesicular mechanism of trans-synaptic communication, potentially in the form of exosomes. To establish the mechanisms for the release of Evi vesicles, we used a dsRNA assay in cultured cells to screen for genes that when down-regulated prevent the release of Evi vesicles. We identified two proteins, Rab11 and Syntaxin 1A (Syx1A), that were required for Evi vesicle release. To determine whether the same mechanisms were used in vivo at the neuromuscular junction, we altered the activity of Rab11 and Syx1A in motoneurons and determined the impact on Evi release. We found that Syx1A, Rab11, and its effector Myosin5 were required for proper Evi vesicle release. Furthermore, ultrastructural analysis of synaptic boutons demonstrated the presence of multivesicular bodies, organelles involved in the production and release of exosomes, and these multivesicular bodies contained Evi. We also used mass spectrometry, electron microscopy, and biochemical techniques to characterize the exosome fraction from cultured cells. Our studies revealed that secreted Evi vesicles show remarkable conservation with exosomes in other systems. In summary, our observations unravel some of the in vivo mechanisms required for Evi vesicle release.

Electrochemical Characterization of Streptavidin-HRP Immobilized on Multiwall Carbon Nanotubes for Biosensor Applications

In this work, we used gold labeled multiwall carbons nanotubes for peroxidase biosensor. The gold labeling on multiwall carbon nanotubes can be achieved with Pressure vapor Deposition (PVD) technique. The obtained carbon nanotubes can be immobilized on gold electrode with the airbrushing technique. The stability and the molecular structure of the labeled multiwall carbon nanotubes were characterized with cyclic voltammetry, impedance spectroscopy and Fourrier Transform Infra-Red spectroscopy (FTIR). It shows a higher conductivity and a good stability in water interface. For streptavidin-HRP immobilization, the labeled gold nanotubes were activated over night with thiol-acid (16 carbons). An activation procedure was achieved with EDC/NHS for HRP-streptavidin immobilization. The development of biosensor for H2O2 detection was observed with the impedance spectroscopy and cyclic voltammetry techniques. This method could be used to determine total H2O2 concentration in the range 4 μM - 160 μM. The results show that the biosensor response depends on the conductivity and the large surface-to-volume ratio attained with multiwall carbon nanotubes. The response of the developed biosensors was reproducible with higher stability.

Microfluidic heavy metal immunoassay based on absorbance measurement

A simple and rapid flow-based multioperation immunoassay for heavy metals using a microfluidic device was developed. The antigen-immobilized microparticles in a sub-channel were introduced as the solid phase into a main channel structures through a channel flow mechanism and packed into a detection area enclosed by dam-like structures in the microfluidic device. A mixture of a heavy metal and a gold nanoparticle-labeled antibody was made to flow toward the corresponding metal through the main channel and make brief contact with the solid phase. A small portion of the free antibody was captured and accumulated on the packed solid phase. The measured absorbance of the gold label was proportional to the free antibody portion and, thus, to the metal concentration. Each of the monoclonal antibodies specific for cadmium-EDTA, chromium-EDTA, or lead-DTPA was applied to the single-channel microfluidic device. Under optimized conditions of flow rate, volume, and antibody concentration, the theoretical (antibody Kd-limited) detection levels of the three heavy metal species were achieved within only 7min. The dynamic range for cadmium, chromium, and lead was 0.57–60.06ppb, 0.03–0.97ppb, and 0.04–5.28ppb, respectively. An integrated microchannel device for simultaneous multiflow was also successfully developed and evaluated. The multiplex cadmium immunoassay of four samples was completed within 8min for a dynamic range of 0.42–37.48ppb. Present microfluidic heavy metal immunoassays satisfied the Japanese environmental standard for cadmium, chromium and, lead, which provided in the soil contamination countermeasures act.

Chemiluminescence-based biosensor for fumonisins quantitative detection in maize samples

A compact portable chemiluminescent biosensor for simple, rapid, and ultrasensitive on-site quantification of fumonisins (fumonisin B1 + fumonisin B2) in maize has been developed. The biosensor integrates a competitive lateral flow immunoassay based on enzyme-catalyzed chemiluminescence detection and a highly sensitive portable charge-coupled device (CCD) camera, employed in a contact imaging configuration. The use of chemiluminescence detection allowed accurate and objective analyte quantification, rather than qualitative or semi-quantitative information usually obtained employing conventional lateral flow immunoassays based on colloidal gold labeling. A limit of detection of 2.5 μg L −1 for fumonisins was achieved, with an analytical working range of 2.5–500 μg L −1 (corresponding to 25–5000 μg kg −1 in maize flour samples, according to the extraction procedure). Total assay time was 25 min, including sample preparation. A simple and convenient extraction procedure, performed by suspending the sample in a buffered solution and rapidly heating to eliminate endogenous peroxidase enzyme activity was employed for maize flour samples analysis, obtaining recoveries in the range 90–115%, when compared with LC–MS/MS analysis. The chemiluminescence immunochromatography-based biosensor is a rapid, low cost portable test suitable for point-of-use applications.

Mapping the distributions and quantifying the labelling intensities of cell compartments by immunoelectron microscopy: progress towards a coherent set of methods

An important tool in cell biology is the combination of immunogold labelling and transmission electron microscopy (TEM) by which target molecules (e.g. antigens) are bound specifically to affinity markers (primary antibodies) and then detected and localised with visualisation probes (e.g. colloidal gold particles bound to protein A). Gold particles are electron-dense, punctate and available in different sizes whilst TEM provides high-resolution images of particles and cell compartments. By virtue of these properties, the combination can be used also to quantify one or more defined targets in cell compartments. During the past decade, new ways of quantifying gold labelling within cells have been devised. Their efficiency and validity rely on sound principles of specimen sampling, event counting and inferential statistics. These include random selection of items at each sampling stage (e.g. specimen blocks, thin sections, microscopical fields), stereological analysis of cell ultrastructure, unbiased particle counting and statistical evaluation of a suitable null hypothesis (no difference in the intensity or pattern of labelling between compartments or groups of cells). The following approaches are possible: (i) A target molecule can be tested for preferential labelling by mapping the localisation of gold particles across a set of compartments. (ii) Data from wild-type and knockdown/knockout control cells can be used to correct raw gold particle counts, estimate specific labelling densities and then test for preferential labeling. (iii) The same antigen can be mapped in two or more groups of cells to test whether there are experimental shifts in compartment labelling patterns. (iv) A variant of this approach uses more than one size of gold particle to test whether or not different antigens colocalise in one or more compartments. (v) In studies involving antigen translocation, absolute numbers of gold particles can be mapped over compartments at specific positions within polarised, oriented or dividing cells. Here, the current state of the art is reviewed and approaches are illustrated with virtual datasets.

The detection of a transgenic soybean biochip using gold label silver stain technology

A method for the rapid detection of transgenic soybean crops based on a combination of gene chip and "gold label silver stain" (GLSS) technologies has been established. To ensure the specificity of this method, the CaMV35S promoter and Nos terminator were selected as probes because they are both exogenous genes that are specific to transgenic soybean plants. The addition of biotin-modified dUTPs to a polymerase chain reaction (PCR) system can produce amplified nucleic acid segments containing biotin. These labeled PCR products then hybridize with specific probes on the chip and are subsequently bound by streptavidin-modified gold nanoparticles (GNPs). Due to the catalytic nature of the GNPs, silver staining can be used to visualize the hybridized probes, which appear as signals in varying shades of gray. The intensity value of the gray signals can be obtained using a general scanner. Silver staining for 10 min was determined to produce the optimal signal-to-noise ratio. In addition, this method was shown to be highly specific and had a detection sensitivity of 288.57 pg/μL.

Ultrastructural analysis of thrombin-induced interaction between human platelets and liposomes carrying fibrinogen γ-chain dodecapeptide as a synthetic platelet substitute

Background The dodecapeptide HHLGGAKQAGDV (H12) in the carboxy-terminus of the fibrinogen γ-chain is a specific binding site of the ligand for platelet GPIIb/IIIa complex. We have evaluated liposomes carrying fibrinogen γ-chain dodecapeptide as a synthetic platelet substitute. Objectives We examined the interaction between human platelets and H12-liposomes during thrombin-induced activation using flow cytometry and electron microscopy (EM). Methods and results After thrombin-activation, a remarkable time-dependent increase in binding of the H12-liposomes to platelets was found by flow cytometry. A large-sized swollen open canalicular system (OCS) was observed in the spheroidal platelets from 60 sec to 5 min after thrombin-activation, but intact H12-liposomes were not evident by conventional EM. Cryoultramicrotomy and immunogold staining with anti-H12 antibody were successful in identifying the liposomes; they appeared as small particles with a unit membrane around 0.2 to 0.4 μm in diameter, and gold labels representing H12 were distributed homogeneously on the surface. Abundant H12-liposomes were localized not only on the surface membrane but also in the lumen of the large-sized swollen OCS in the platelets at 60 sec after thrombin-activation. The formation of the large-sized swollen OCS was inhibited by pre-incubation with unbound H12, EDTA or anti-GPIIb/IIIa antibody. In thrombin-induced platelet aggregates we observed electron-transparent areas between adherent platelets, in which abundant H12-liposomes were distributed. Conclusions We demonstrate morphologically that H12-liposomes bind to thrombin-activated platelets and accumulate between adherent platelets like fibrinogen, leading to large-scale aggregation.

Pretreatment-free immunochromatographic assay for the detection of streptomycin and its application to the control of milk and dairy products

A rapid pretreatment-free immunochromatographic assay was developed for the control of the streptomycin (STR) content in milk and dairy products. The assay is based on the competition between an immobilized STR–protein conjugate and STR in a sample to be tested for the binding to monoclonal anti-STR antibodies conjugated to colloidal gold during the flow of the sample along a membrane strip with immobilized reactants. It is possible to improve the cut-off level of positive and negative samples distinguished by a change in the molar STR to protein ratio in the immobilized conjugate. The cut-off level (500 ng mL −1) thus achieved corresponds to the stated MRL of STR in milk and dairy products. For STR concentrations in the range of 16–250 ng mL −1 its content can be quantitatively measured based on the degree of binding of a colloidal gold label in the test strip zone with the immobilized STR–protein conjugate. The duration of the assay is 10 min. The selected sizes of membrane pores and colloidal gold particles allow the assay to be carried out at room temperature without additional reactants and pretreatment. The applicability of the assay for milk, whole milk, sour clotted milk, and kefir with different fat content (from 0.5% to 6%) was confirmed. The results of quantitative immunochromatographic assay show good correlation with traditional ELISA ( r was equal to 0.935 and 0.940 for the series tested).

Quantifying immunogold localization on electron microscopic thin sections: a compendium of new approaches for plant cell biologists

A review is presented of recently developed methods for quantifying electron microscopical thin sections on which colloidal gold-labelled markers are used to identify and localize interesting molecules. These efficient methods rely on sound principles of random sampling, event counting, and statistical evaluation. Distributions of immunogold particles across cellular compartments can be compared within and between experimental groups. They can also be used to test for co-localization in multilabelling studies involving two or more sizes of gold particle. To test for preferential labelling of compartments, observed and expected gold particle distributions are compared by χ(2) analysis. Efficient estimators of gold labelling intensity [labelling density (LD) and/or relative labelling index (RLI)] are used to analyse volume-occupying compartments (e.g. Golgi vesicles) and/or surface-occupying compartments (e.g. cell membranes). Compartment size is estimated by counting chance events after randomly superimposing test lattices of points and/or line probes. RLI=1 when there is random labelling and RLI >1 when there is preferential labelling. Between-group comparisons do not require information about compartment size but, instead, raw gold particle counts in different groups are compared by combining χ(2) and contingency table analyses. These tests may also be used to assess co-distribution of different sized gold particles in compartments. Testing for co-labelling involves identifying sets of compartmental profiles that are unlabelled and labelled for one or both of two gold marker sizes. Numbers of profiles in each labelling set are compared by contingency table analysis and χ(2) analysis or Fisher's exact probability test. The various methods are illustrated with worked examples based on empirical and synthetic data and will be of practical benefit to those applying single or multiple immunogold labelling in their research.

Neuroglial and synaptic rearrangements associated with photic entrainment of the circadian clock in the suprachiasmatic nucleus

AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL In the published paper of Girardet et al. (2010), the graphs comparing the mean synaptic innervation of VIP dendrites by GABAergic terminals (GABA+) and non-GABAergic terminals (GABA−) have been inverted (Fig. 4E). The correct data were those that had been described in the text (no day-night variations vs 62% increase in the respective synaptic density of GABAergic terminals and non-GABAergic terminals. The authors apologize for this error. Figure 4Open in figure viewerPowerPoint Quantitative electron-microscopic evaluation of the effect of time on the density of GABAergic and non-GABAergic synapses onto SCN VIP dendrites. Data were obtained using the unbiaised dissector method after analysis of pairs of serial ultrathin sections. According to the method (Sterio, 1984), only the synapses in the first ‘reference’ section which disappeared in the second section (‘look-up’ section) were counted. The GABAergic endings were gold-labelled with a polyclonal anti-GABA antiserum (kindly provided by Dr P. Somogyi; see Hodgson et al., 1985), according to a post-embedding procedure (Andréet al., 1987). The gold labelling was carried out on SCN sections containing peroxidase-stained VIP dendrites (see Becquet et al., 2008). (A–D) Examples of pairs of GABA–VIP double-labelled ultrathin sections. The synapse shown in A (filled arrowheads) between a GABAergic (gold-labelled) terminal and a VIP (peroxidase-stained) dendrite is no longer visible in B. In C and D, the VIP-positive dendrite shares a synapse with a GABA-positive ending which is visible on the two paired sections (filled arrowheads). It receives another synapse from a nonimmunoreactive ending (empty arrowheads) which has disappeared on section D. (E) Day–night variations in the mean synaptic innervation of VIP dendrites by GABAergic terminals (GABA+) and non-GABAergic terminals (GABA−). Data are related to the volume of the postsynaptic partner. The quantitative analysis was carried out after sampling of 127 VIP-stained dendritic profiles in rats killed at ZT02 (daytime, n = 4 rats) and of 111 such profiles in rats killed at ZT18 (nighttime, n = 4 rats). It involved 139 serially sectioned terminals in contact with at least one VIP dendrite at ZT02, among which 98 were GABA-positive, and 124 such terminals including 82 GABA-positive ones at ZT18. The mean numbers of synapses (Nv) per μm3 dendrite was estimated according to the equation Nv = ΣQ−/Vdis, where ΣQ− represents the total number of synapses present in the ‘reference’ section which disappeared in the ‘look-up’ section, and Vdis the dissector volume, derived from the area of the dendrites (as measured using the NIH ImageJ image processing software) multiplied by the section thickness. *P < 0.05 with the two-way Kruskal–Wallis anova test (Statistica, Statsoft). Scale bars, 0.5 μm. References André, D., Vuillon-Cacciuttolo, G. & Bosler, O. (1987) GABA nerve endings in the rat red nucleus combined detection with serotonin terminals using dual immunocytochemistry. Neuroscience, 23, 1095– 1102. Becquet, D., Girardet, C., Guillaumond, F., François-Bellan, A. & Bosler, O. (2008) Ultrastructural plasticity in the rat suprachiasmatic nucleus. Possible involvement in clock entrainment. Glia, 56, 294– 305. Girardet, C., Becquet, D., Blanchard, M.-P., François-Bellan, A.-M. & Bosler, O. (2010) Neuroglial and synaptic rearrangements associated with photic entrainment of the circadian clock in the suprachiasmatic nucleus. Eur. J. Neurosci., 32, 2133– 2142. Hodgson, A.J., Penke, B., Erdei, A., Chubb, I.W. & Somogyi, P. (1985) Antisera to gamma-aminobutyric acid. I. Production and characterization using a new model system. J. Histochem. Cytochem., 33, 229– 239. Sterio, D.C. (1984) The unbiased estimation of number and sizes of arbitrary particles using the disector. J. Microsc., 134, 127– 136. Volume33, Issue8April 2011Pages 1561-1561 FiguresReferencesRelatedInformation

Gold nanoparticle labeling of cells is a sensitive method to investigate cell distribution and migration in animal models of human disease

The ability to track cells in small-animal models of human disease is important because it gives the potential to improve our understanding of the processes of disease progression as well as our understanding of the therapeutic effects of interventions. In this study gold nanoparticles have been used as a permanent marker of implanted normal and malignant cell grafts in combination with a suitable x-ray apparatus. Using x-ray computed tomography the micrometric three-dimensional distribution of these marked cells could be displayed with penetration depth, high cell sensitivity and high spatial resolution in rodent models of human diseases. In principle the method allows quantification of cell numbers at any anatomical location over time in small animals. From the Clinical Editor In this paper, a gold nanoparticle based cell labeling method is presented for in vivo cell tracking via micro-CT. Although a tumor model is shown in this pilot experiment, the method can theoretically be utilized in cell labeling experiments of any kind.

Does the Gold Standard label hold its promise in delivering higher Sustainable Development benefits? A multi-criteria comparison of CDM projects

The Clean Development Mechanism (CDM) has a twin objective: to help developed countries reduce GHG emissions, and to support developing countries in achieving Sustainable Development (SD). As a response to the widespread criticism of the CDM's unsatisfactory SD record, initiatives have developed premium labels like the Gold Standard, which applies two additional ‘screens’ to filter CDM projects for higher SD benefits. In order to determine whether Gold Standard projects can be associated with higher local SD benefits, this paper evaluates the potential benefits of 48 CDM projects using a multi-criteria method and building on existing work. The 18 evaluated Gold Standard projects are compared to a ‘representative portfolio’ of 30 unlabeled CDM projects in order to capture the ‘full’ effect of the additional Gold Standard requirements, which is further decomposed into the two ‘screen’ effects. The results suggest that Gold Standard Certified Emission Reductions can be associated with higher potential local SD benefits when compared to the ‘representative portfolio’ of unlabeled CDM projects, while the comparison of projects of the same type remains inconclusive. The results support previous findings showing that renewable energy projects may deliver comparatively high SD benefits.