[The auxiliary diagnostic value of ECP and MPO expression in nasal secretions in different types of rhinitis].

Objective:To evaluate the expression of eosinophil cationic protein and myeloperoxidase in nasal secretions in different types of rhinitis, and to explore their values in the differential diagnosis of different types of rhinitis. Methods:Six hundred and eighty-four subjects were selected, including 62 subjects in the acute rhinitis group, 378 subjects in the allergic rhinitis group, 94 subjects in the vasomotor rhinitis group, 70 subjects in the eosinophilic non-allergic rhinitis group, and 80 subjects in the control group. Nasal secretion samples were collected from the five groups, and the percentages of inflammatory cells were counted by Rachel's staining, and the expression of ECP/MPO was detected by colloidal gold assay. The correlation between the clinical diagnosis, the inflammatory cells in the nasal secretions and the expression of ECP/MPO was analyzed. Results:Nasal cytological smears showed that compared with the control group, the percentage of eosinophils in the AR and NARES groups were significantly higher （P<0.05）, while the percentage of neutrophils was not different （P>0.05）; the percentage of neutrophils was significantly higher in the acute rhinitis group compared with the control group （P<0.05）, while the percentage of eosinophils was not statistically different （P>0.05）; in vasomotor rhinitis group, the eosinophils and neutrophils were not statistically different compared with the control group（P> 0.05）. The colloidal gold results showed that there were differences in the expression of ECP/MPO in different types of rhinitis, among which 49 cases （79.0%） in the acute rhinitis group expressed ECP+/MPO+; 267 cases （70.6%） in the AR group and 56 cases （75.7%） in the NARES group expressed ECP+/MPO-; 80 cases （85.1%） in the vasomotor rhinitis group and 69 cases （86.3%） in the control group expressed ECP-/MPO-. Conclusion:The differences in ECP and MPO expression between different types of rhinitis have certain reference value for the differential diagnosis of different types of rhinitis and the selection of treatment programs.

A Rapid Intraoperative Parathyroid Hormone Assay Based on the Immune Colloidal Gold Technique for Parathyroid Identification in Thyroid Surgery.

Objective: A novel immunochromatographic test strip method was developed to detect tissue parathyroid hormone (PTH) using the immune colloidal gold technique (ICGT). The accuracy and application value of this method for intraoperative parathyroid identification were evaluated. Methods: Serum samples were collected to measure PTH by both ICGT and electrochemiluminescence immunoassay (ECLIA). Patients who underwent unilateral and total thyroidectomy were enrolled to evaluate the feasibility and clinical efficacy of rapid intraoperative identification of parathyroid glands via PTH determination using ICGT. Two sample preparation methods, fine needle aspiration (FNA) and tissue block homogenate (TBH), were used for PTH-ICGT analysis. Results: Bablok analysis showed a linear relationship between the serum PTH measurements obtained by ICGT and ECLIA. Non-parathyroid tissues had much lower PTH concentrations (14.8 ± 2.1 pg/ml, n = 97) detected by ICGT, compared to the parathyroid gland tissues (955.3 ± 16.1 pg/ml, n = 79; P < 0.0001), With biopsy results as the standard, ICGT showed higher diagnosis rates as compared with direct visual inspection, for identifying both parathyroid glands (97.4 vs. 78.2%) and non-parathyroid tissues (100 vs. 68.9%). The cut-off values for parathyroid identification by FNA and TBH methods were 63.99 and 136.30 pg/ml, respectively. The detection time was 2 min by TBH method for in vitro tissue detection and 6 min by FNA method for in situ tissue detection, both of which were faster than traditional intraoperative cryopathological examination (usually >30 min). Intraoperative application of ICGT method was associated with higher postoperative serum calcium and blood PTH levels at 1 and 3 months as well as a lower incidence of postoperative transient hypocalcemia, as compared with direct visual inspection. Conclusion: PTH-ICGT assay shows high potential as a rapid, novel alternative for intraoperative parathyroid identification.

Detecting Gold-Labeled Cells.

Colloidal gold particles bind tightly but not covalently to proteins at pH values that are around the protein's pI. Colloidal gold particles conjugated with a wide range of anti-immunoglobulin antibodies, Protein A, or streptavidin are available commercially. Gold labels were developed originally for electron microscopic studies, but they also work well at the level of the light microscope. They give higher resolution than enzyme-based methods and avoid the problems of substrate preparation and endogenous enzyme activity. Until recently, the gold labels lacked sensitivity at the level of light microscopy, but the recent development of the photochemical silver method of amplification, described here, has overcome this problem. Unamplified gold labels can be detected under the light microscope using bright-field illumination in which the label ranges from pale pink to deep red, depending on the strength of the reaction. Nomarski differential interference contrast microscopy makes the label appear dark red to black. With the silver enhancement method, the gold particles become coated in metallic silver and yield a black-brown label, best visualized by bright-field optics. Gold labeling methods are compatible with many histochemical stains. Gold labeling reactions are very readily controlled, because the appearance of staining can be monitored directly and continuously under the microscope.

Bordered pits in xylem of vesselless angiosperms and their possible misinterpretation as perforation plates.

Vesselless wood represents a rare phenomenon within the angiosperms, characterizing Amborellaceae, Trochodendraceae and Winteraceae. Anatomical observations of bordered pits and their pit membranes based on light, scanning and transmission electron microscopy (SEM and TEM) are required to understand functional questions surrounding vesselless angiosperms and the potential occurrence of cryptic vessels. Interconduit pit membranes in 11 vesselless species showed a similar ultrastructure as mesophytic vessel-bearing angiosperms, with a mean thickness of 245 nm (± 53, SD; n = six species). Shrunken, damaged and aspirated pit membranes, which were 52% thinner than pit membranes in fresh samples (n = four species), occurred in all dried-and-rehydrated samples, and in fresh latewood of Tetracentron sinense and Trochodendron aralioides. SEM demonstrated that shrunken pit membranes showed artificially enlarged, > 100 nm wide pores. Moreover, perfusion experiments with stem segments of Drimys winteri showed that 20 and 50 nm colloidal gold particles only passed through 2 cm long dried-and-rehydrated segments, but not through similar sized fresh ones. These results indicate that pit membrane shrinkage is irreversible and associated with a considerable increase in pore size. Moreover, our findings suggest that pit membrane damage, which may occur in planta, could explain earlier records of vessels in vesselless angiosperms.

Current state and prospects of the phytosynthesized colloidal gold nanoparticles and their applications in cancer theranostics.

The design, development, and biomedical applications of phytochemical-based green synthesis of biocompatible colloidal gold nanoparticles (AuNPs) are becoming an emerging field due to several advantages (safer, eco-friendly, simple, fast, energy efficient, low-cost, and less toxic) over conventional chemical synthetic procedures. Biosynthesized colloidal gold nanoparticles are remarkably attractive in several biomedical applications including cancer theranostics due to small size, unusual physico-chemical properties, facile surface modification, high biocompatibility, and numerous other advantages. Of late, several researchers have investigated the biosynthesis and prospective applications (diagnostics, imaging, drug delivery, and cancer therapeutics) of AuNPs in health care and medicine. However, not a single review article is available in the literature that demonstrates the anti-cancer potential of biosynthesized colloidal AuNPs with detailed mechanistic study. In the present review article, we for the first time discuss the biointerface of colloidal AuNPs, plants, and cancer mainly (i) comprehensive mechanistic aspects of phytochemical-based synthesis of AuNPs; (ii) proposed anti-cancer mechanisms along with biomedical applications in diagnostics, imaging, and drug delivery; and (iii) key challenges for biogenic AuNPs as future cancer nanomedicine.

Synaptic contact between median preoptic neurons and subfornical organ neurons projecting to the paraventricular hypothalamic nucleus.

It is known that the median preoptic nucleus (POMe) sends dense projections to the subfornical organ (SFO). However, the functional significance of these projections have not been well discussed. In this electron microscopic study, we investigated the presence of synapses between POMe-derived axon terminals and SFO neurons that project to the paraventricular hypothalamic nucleus (PVN). After injection of a retrograde tracer, wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex, into the PVN, many labeled neurons were found in the SFO. In contrast, after injection of an anterograde tracer, biotinylated dextran amine, in the POMe, abundant labeled axon varicosities were observed in the SFO. Using electron microscopy, synapses were identified between retrogradely labeled dendrites and cell bodies, and anterogradely labeled axon terminals, indicating that POMe neurons innervate SFO neurons projecting to the PVN. The possibility that POMe neurons play multiple roles in the neuronal circuit responsible for vasopressin release and/or cardiovascular regulation is also discussed.

Specific and Sensitive Isothermal Electrochemical Biosensor for Plant Pathogen DNA Detection with Colloidal Gold Nanoparticles as Probes.

Developing quick and sensitive molecular diagnostics for plant pathogen detection is challenging. Herein, a nanoparticle based electrochemical biosensor was developed for rapid and sensitive detection of plant pathogen DNA on disposable screen-printed carbon electrodes. This 60 min assay relied on the rapid isothermal amplification of target pathogen DNA sequences by recombinase polymerase amplification (RPA) followed by gold nanoparticle-based electrochemical assessment with differential pulse voltammetry (DPV). Our method was 10,000 times more sensitive than conventional polymerase chain reaction (PCR)/gel electrophoresis and could readily identify P. syringae infected plant samples even before the disease symptoms were visible. On the basis of the speed, sensitivity, simplicity and portability of the approach, we believe the method has potential as a rapid disease management solution for applications in agriculture diagnostics.

Development of a Colloidal Gold-Based Immunochromatographic Strip for Rapid Detection of Klebsiella pneumoniae Serotypes K1 and K2.

In this study, a novel colloidal gold-based immunochromatographic strip (ICS) containing anti-Klebsiella pneumoniae capsular polysaccharide polyclonal antibodies was developed to specifically detect K. pneumoniae serotypes K1 and K2. Capsular polysaccharide K1 and K2 antigens were first used to produce polyclonal anti-K1 and anti-K2 antibodies. Reference strains with different serotypes, nontypeable K. pneumoniae strains, and other bacterial species were then used to assess the sensitivity and specificity of these test strips. The detection limit was found to be 105 CFU, and the ICSs were stable for 6 months when stored at room temperature. No false-positive or false-negative results were observed, and equivalent results were obtained compared to those of more conventional test methods, such as PCR or serum agglutination. In conclusion, the ICS developed here requires no technical expertise and allows for the specific, rapid, and simultaneous detection of K. pneumoniae serotypes K1 and K2.

Development of a colloidal gold immunochromatographic strip for the rapid detection of soft-shelled turtle systemic septicemia spherical virus.

A colloidal gold immunochromatographic strip (ICS) test based on a competitive format was developed for the rapid detection of soft-shelled turtle systemic septicemia spherical virus (STSSSV) in turtle and fecal samples. Specific egg yolk antibodies (IgY) against STSSSV were labeled with colloidal gold and used as probes in the one-step test strip. Antigen (STSSSV) and goat anti-chicken IgY were drawn on the nitrocellulose membrane as the test line and control line, respectively. When STSSSV standard samples (0-100µg/mL) were detected by the strips, the visual limit of detection (LOD) was found to be 50.0µg/mL. The ICS test showed high stability; the strips were stable for at least 3 months at 4°C without significant loss of activity. There was no obvious cross-reactivity with other aquatic pathogens. The assay can be performed within 5-10min. Analysis of STSSSV in turtle samples revealed that data obtained from the ICS test were in a good agreement with those obtained by ELISA. The positive results of fecal samples suggested that this method could be used to detect STSSSV while protecting the animals' welfare. The ICS assay does not need specialized equipment or a technician and can be used as a reliable, rapid, cost-effective and convenient qualitative tool for on-site diagnosis.

Nanoparticle accumulation and transcytosis in brain endothelial cell layers.

The blood-brain barrier (BBB) is a selective barrier, which controls and limits access to the central nervous system (CNS). The selectivity of the BBB relies on specialized characteristics of the endothelial cells that line the microvasculature, including the expression of intercellular tight junctions, which limit paracellular permeability. Several reports suggest that nanoparticles have a unique capacity to cross the BBB. However, direct evidence of nanoparticle transcytosis is difficult to obtain, and we found that typical transport studies present several limitations when applied to nanoparticles. In order to investigate the capacity of nanoparticles to access and transport across the BBB, several different nanomaterials, including silica, titania and albumin- or transferrin-conjugated gold nanoparticles of different sizes, were exposed to a human in vitro BBB model of endothelial hCMEC/D3 cells. Extensive transmission electron microscopy imaging was applied in order to describe nanoparticle endocytosis and typical intracellular localisation, as well as to look for evidence of eventual transcytosis. Our results show that all of the nanoparticles were internalised, to different extents, by the BBB model and accumulated along the endo-lysosomal pathway. Rare events suggestive of nanoparticle transcytosis were also observed for several of the tested materials.

A rapid and highly sensitive protocol for the detection of Escherichia coli O157:H7 based on immunochromatography assay combined with the enrichment technique of immunomagnetic nanoparticles.

BACKGROUND: Escherichia coli O157:H7 (E. coli O157:H7) is an important pathogenic bacterium that threatens human health. A rapid, simple, highly sensitive, and specific method for the detection of E. coli O157:H7 is necessary. METHODS: In the present study, immunomagnetic nanoparticles (IMPs) were prepared with nanopure iron as the core, coated with E. coli O157:H7 polyclonal antibodies. These IMPs were used in combination with immunochromatographic assay (ICA) and used to establish highly sensitive and rapid kits (IMPs+ICA) to detect E. coli O157:H7. The kits were then used to detect E. coli O157:H7 in 150 food samples and were compared with conventional ICA to evaluate their efficacy. RESULTS: The average diameter of IMPs was 56 nm and the amount of adsorbed antibodies was 106.0 µg/mg. The sensitivity of ICA and IMPs+ICA was 10(5) colony-forming units/mL and 10(3) CFUs/mL, respectively, for purified E. coli O157:H7 solution. The sensitivity of IMPs+ICA was increased by two orders, and its specificity was similar to ICA. CONCLUSION: The kits have the potential to offer important social and economic benefits in the screening, monitoring, and control of food safety.

Development of a serotype colloidal gold strip using monoclonal antibody for rapid detection type Asia1 foot-and-mouth disease.

BACKGROUND: In this study, we developed a rapid, one step colloid gold strip (CGS) capable of specifically detecting type Asia1 foot-and-mouth disease virus (FMDV). We have produced two monoclonal antibodies (mAb) to type Asia1 FMD (named 1B8 and 5E2). On the test strip, the purified 1B8 labelled with the colloidal gold was used as the detector, and the purified 5E2 and goat anti-mouse antibodies were wrapped onto nitrocellulose (NC) membranes as the test and the control line, respectively. The rapid colloidal gold stereotype diagnostic strip was housed in a plastic case. RESULTS: In specificity and sensitivity assay, there was no cross-reaction of the antigen with the other type of FMD and SVDV. The detection sensitivity was found to be as high as 10(-5) dilution of Asia1/JSL/05 (1 × 10(7.2)TCID(50)/50 µL). There was excellent agreement between the results obtained by CGS and reverse indirect hemagglutination assay (RIHA), and the agreement can reach to 98.75%. CONCLUSION: We developed colloidal gold strips that have good qualities and does not require specialized equipment or technicians. This method provided a feasible, convenient, rapid, and effective for detecting type Asia1 FMDV in the fields.

Macrophage scavenger receptor A mediates the uptake of gold colloids by macrophages in vitro.

AIMS: While numerous studies have reported on nanoparticle uptake by phagocytic cells, the mechanisms of this uptake are poorly understood. A metastudy of research focusing on biological particulate matter has postulated that nanoparticles cannot be phagocytosed and therefore must enter cells via pinocytosis. The purpose of this study was to identify the route(s) of uptake of gold nanoparticles in vitro and to determine if these route(s) depend on particle size. MATERIALS & METHODS: The parent RAW264.7 cell line and its derivatives, transduced with a virus carrying siRNA to macrophage scavenger receptor A, were used as model phagocytes. Citrate-stabilized gold colloids were used as model nanoparticles. We used chemical inhibitors known to interfere with specific routes of particulate uptake. We developed multifocal light microscopy methods including multifocal stack analysis with NIH ImageJ software to analyze cell uptake. RESULTS: Irrespective of size, gold nanoparticles are internalized by macrophages via multiple routes, including both phagocytosis and pinocytosis. If either route was blocked, the particles entered cells via the other route. CONCLUSION: Gold nanoparticles with hydrodynamic sizes below 100 nm can be phagocytosed. Phagocytosis of anionic gold colloids by RAW264.7 cells is mediated by macrophage scavenger receptor A.

Filter-feeding bivalves store and biodeposit colloidally stable gold nanoparticles.

Nanoparticles resistant to salt-induced aggregation are continually being developed for biomedical and industrial applications. Because of their colloidal stability these functionalized nanoparticles are anticipated to be persistent aquatic contaminants. Here, we show that Corbicula fluminea, a globally distributed clam that is a known sentinel of aquatic ecosystem contamination, can uptake and biodeposit bovine serum albumin (BSA) stabilized gold nanoparticles. Nanoparticle clearance rates from suspension were dictated by diameter and concentration, with the largest particles cleared most quickly on a mass basis. Particle capture facilitates size-selective 'biopurification' of particle suspensions with nanoscale resolution. Nanoparticles were retained either within the clam digestive tract or excreted in feces. Our results suggest that biotransformation and biodeposition will play a significant role in the fate and transport of persistent nanoparticles in aquatic systems.

Development of an active targeting liposome encapsulated with high-density colloidal gold for transmission electron microscopy.

Active targeting of the liposome is an attractive strategy for drug delivery and in vivo bio-imaging. We previously reported the specific accumulation of Sialyl Lewis X (SLX) liposome to inflamed tissue in arthritic model mice or tumor-bearing mice. SLX-liposome encapsulation with fluorescent substances allows for the visualization of these liposomes by the time-dependent transvascular accumulation of fluorescent signals in the histological sections. In the present study, we developed a new SLX-liposome encapsulated with colloidal gold for transmission electron microscopic observation. We herein describe the characterization of the colloidal gold-loaded SLX-liposomes and demonstrate its specific targeting to the endothelial cells of tumor blood vessels in tumor-bearing mice.

V1 interpatch projections to v2 thick stripes and pale stripes.

Cytochrome oxidase (CO) reveals two compartments in V1 (patches and interpatches) and three compartments in V2 (thin, pale, and thick stripes). Previously, it was shown that thin stripes receive input predominantly from patches. Here we examined the projections to thick and pale stripes in macaques, revealed by retrograde tracer injections. After thick stripe injection, cells were distributed in layer 2/3 (67%), layer 4A (7%), layer 4B (23%), and layer 5/6 (2%). Except in layer 5/6, cells were concentrated in interpatches, with a stronger bias in layer 2/3 (84%) than in layer 4B (75%). After pale stripe injection, cells were found in layer 2/3 (87%), layer 4A (2%), layer 4B (10%), and layer 5/6 (2%). As for thick stripes, cells were located preferentially in interpatches in layer 2/3 (84%) and layer 4B (72%) but not in layer 5/6. Thick stripes received a higher proportion of their input from layer 4B, compared with pale stripes, consistent with reports that thick stripe neurons exhibit a pronounced layer 4B influence. This difference aside, both stripe types receive similar inputs from V1, at least in terms of cortical layer and CO compartment. This finding was bolstered by injecting different tracers into pale and thick stripes; 10-27% of cells were double labeled, with most located in interpatches. These results suggest that the distinctive receptive field properties of neurons in thick and pale stripes are generated by local V2 circuits, or by other specific projections, rather than by differing sources of laminar and compartmental input from V1.

Binding of beta-secretase to a peptide inhibitor-carrying SAM.

A novel disulfide, which carried a specific inhibitor for beta-secretase (KMI360) at both ends, was prepared by the coupling of 11,11'-dithiobisundecanoic acid (DTUA) with the inhibitor. The compound obtained (DTUA-KMI360) formed a self-assembled monolayer (SAM) on a gold electrode as proven by cyclic voltammetry (CV) using hydroquinone as a probe. Furthermore, DTUA-KMI360 could be accumulated as a SAM on a gold colloid deposited on a glass plate (Au colloid-glass chip) as proven by both the red-shift and the increase in absorbance of the gold colloid corresponding to localized surface plasmon resonance (LSPR). When the SAM-modified Au colloid-glass chip was immersed in a solution of aspartyl proteases, pepsin and beta-secretase, the absorbance of the chip at 550nm corresponding to LSPR of the gold colloid further increased and was slightly red-shifted, whereas coexistence of a free inhibitor obstructed these phenomena. Adsorption of the enzymes was promoted by the incorporation of a zwitterionic group into the SAM, while non-specific adsorption to the mixed SAM was significantly reduced. The optimal ratio of omega-zwitterionic alkanethiol, 3-[(6-mercaptohexyl)-N,N-dimethylamino]propane-1-sulfonic acid (C(6)-SPB), and DTUA-KMI360 in the SAM for the binding of enzymes was found to be DTUA-KMI360:C(6)-SPB=1:11 using polarization modulation infrared reflection absorption spectroscopy (PM-IR-RAS). From increasing profiles of absorbance of the Au colloid-glass chip, the association constant (K(assoc)) for pepsin with the inhibitor on the SAM was determined, whereas that for beta-secretase could not be due to the strong binding of the enzyme to the inhibitor, resulting in the absence of the dissociation process. The results suggested that the SAM of the enzyme inhibitor can be used for both investigation of enzymes and removal of target enzymes from biological fluids.

Localization of anionic constituents in mast cell granules of brachymorphic (bm/bm) mice by using avidin-conjugated colloidal gold.

We used the egg avidin gold complex as a polycationic probe for the localization of negatively charged sites in the secretory granules of mouse mast cells. We compared the binding of this reagent to mast cell granules in wild-type mice and in congenic brachymorphic mice in which mast cell secretory granules contained undersulfated proteoglycans. We localized anionic sites by post-embedding labeling of thin sections of mouse skin and tongue tissues fixed in Karnovsky's fixative and OsO(4) and embedded in Araldite. Transmission electron microscopy revealed that the mast cell granules of bm/bm mice had a lower optical density than those of wild-type mice (P<0.001) and a lower avidin gold binding density (by approximately 50%, P<0.001). The latter result provided additional evidence that the contents of mast cell granules in bm/bm mice were less highly sulfated than in those of wild-type mice. In both wild-type and bm/bm mast cells, the distribution of granule equivalent volumes was multimodal, but the unit granule volume was approximately 19% lower in bm/bm cells than in wild-type cells (P<0.05). Thus, bm/bm mast cells develop secretory granules that differ from those of wild-type mice in exhibiting a lower optical density and slightly smaller unit granules, however the processes that contribute to granule maturation and granule-granule fusion in mast cells are operative in bm/bm cells.

Immunogold-silver staining-on-a-chip biosensor based on cross-flow chromatography.

Immunogold-silver staining (IGSS) was adopted in cross-flow chromatographic analysis in which immunological reactions and silver intensification were sequentially conducted in the vertical and horizontal directions, respectively. Factors controlling the performance, except the silver substrate solution, were optimized to increase the signal-to-background ratio in measurements of cardiac troponin I as a model analyte. In generating the signal, the size of colloidal gold catalyst was critical; the smallest size (5-nm diameter) in the selected range yielded the highest colorimetric signal. To maintain the low background, two processes, blocking the remaining surfaces of membrane after antibody immobilization and washing the residual tracer after immunological reaction, were necessary. Self-nucleation of silver ions also caused a background signal and was controlled to some degree by decreasing the hydrodynamic force that arose when the substrate solution was supplied in the horizontal direction. Finally, a new chip (IGSS-on-a-chip; IOC) that allowed for convenient, efficient IGSS was produced by injection molding of plastic. This method enhanced the detection capability by 51-fold compared to the conventional rapid test kit using 30nm-sized colloidal gold as the tracer. The IOC biosensor results also showed that silver intensification yield via cross flow after immunological reaction was 19% higher than that by traditional incubation.

Inhibition of selectin binding by colloidal gold with functionalized shells.

Gold colloids with terminally functionalized sulfated thiol shells were found to bind to P- and L-selectins with IC(50) values in the picomolar range; branched acyclic epitopes show the highest affinity, whereas a sulfated carbohydrate mimetic provides the best selectivity.