Estimating Neospora caninum prevalence in wildlife populations using Bayesian inference

Prevalence of disease in wildlife populations, which is necessary for developing disease models and conducting epidemiologic analyses, is often understudied. Laboratory tests used to screen for diseases in wildlife populations often are validated only for domestic animals. Consequently, the use of these tests for wildlife populations may lead to inaccurate estimates of disease prevalence. We demonstrate the use of Bayesian latent class analysis (LCA) in determining the specificity and sensitivity of a competitive enzyme‐linked immunosorbent assay (cELISA; VMRD^®, Inc.) serologic test used to identify exposure to Neospora caninum (hereafter N. caninum) in three wildlife populations in southeastern Ohio, USA. True prevalence of N. caninum exposure in these populations was estimated to range from 0.1% to 3.1% in American bison (Bison bison), 51.0% to 53.8% in Père David's deer (Elaphurus davidianus), and 40.0% to 45.9% in white‐tailed deer (Odocoileus virginianus). The accuracy of the cELISA in American bison and Père David's deer was estimated to be close to the 96% sensitivity and 99% specificity reported by the manufacturer. Sensitivity in white‐tailed deer, however, ranged from 78.9% to 99.9%. Apparent prevalence of N. caninum from the test results is not equal to the true prevalence in white‐tailed deer and Père David's deer populations. Even when these species inhabit the same community, the true prevalence in the two deer populations differed from the true prevalence in the American bison population. Variances in prevalence for some species suggest differences in the epidemiology of N. caninum for these colocated populations. Bayesian LCA methods could be used as in this example to overcome some of the constraints on validating tests in wildlife species. The ability to accurately evaluate disease status and prevalence in a population improves our understanding of the epidemiology of multihost pathogen systems at the community level.     

New bis(thiosemicarbazonate) gold(III) complexes inhibit HIV replication at cytostatic concentrations: potential for incorporation into virostatic cocktails

Four bis(thiosemicarbazonate)gold(III) complexes (1-4) with a general formula [Au(L)]Cl {L = L1, glyoxal-bis(N⁴-methylthiosemicarbazone); L2, glyoxal-bis(N⁴-ethylthiosemicarbazone); L3, diacetyl-bis(N⁴-methylthiosemicarbazone); L4, diacetyl-bis(N⁴-ethylthiosemicarbazone)} were synthesised and screened for activity against the human immunodeficiency virus (HIV). Complexes 1-4 were characterised using ¹H-NMR and IR spectroscopy; and their purity established by micronanalysis. Complex 3 inhibited viral infection of TZM-bl cells by 98% (IC₅₀ = 6.8 ± 0.6 μM) at a non toxic concentration of 12.5 μM while complex 4 inhibited infection of these cells by 72 and 98% (IC₅₀ = 5.3 ± 0.4 μM) at concentrations of 6.25 and 12.5 μM respectively. The mechanism of inhibition of infection in TZM-bl cells is presumably as a result of the cytostatic or anti-proliferative activity that was observed for complex 4 in real time cell electronic sensing (RT-CES) and carboxyflourescein succinimidyl ester (CFSE) analysis. Treatment of T lymphocytes from HIV infected individuals with 4 decreased CD4+ T cell expression (p = 0.0049) as demonstrated by multi-parametric flow cytometry without suppressing cytokine production. None of the ligands (L1-L4) demonstrated anti-viral activity, supporting the importance of metal (gold) complexation in these potential drugs. Complexes 3 and 4 were shown to have ideal lipophilicity values that were similar when shake flask (0.97 ± 0.5 and 2.42 ± 0.6) and in silico prediction (0.8 and 1.5) methods were compared. The activity and drug-like properties of complexes 3 and 4 suggests that these novel metal-based compounds could be combined with virus inhibitory drugs to work as cytostatic agents in the emerging class of anti-HIV drugs known as virostatics.     

Affinity immunosensor for milk progesterone: identification of critical parameters

The effects of several experimental parameters on the performance characteristics of a competitive-type immunochromatographic assay of milk progesterone were studied. Increasing the size of the colloidal gold particles used as a label increased both maximal signal obtained and sensitivity of the assay measured as slope of the progesterone standard curve. The concentration of the antibody used to prepare the detection zone was found to be a critical factor, in that low concentrations of antibody resulted in a poor sensitivity. The compatibilities of various buffer systems with the assay were studied. The assay worked well with buffers having a broad pH range of 4·5–8·5.     

Elucidation of structural and functional properties of albumin bound to gold nanoparticles

Nanoparticle–albumin complexes are being designed for targeted drug delivery and imaging. However, the changes in the functional properties of albumin due to adsorption on nanoparticles remain elusive. Thus, the objective of this work was to elucidate the structural and functional properties of human and bovine serum albumin bound to negatively charged gold nanoparticles (GNPs). Fluorescence data demonstrated static quenching of albumin by GNP with the quenching of buried as well as surface tryptophan in BSA. The binding process was enthalpy and entropy-driven in HSA and BSA, respectively. At lower concentrations of GNP there was a higher affinity for tryptophan, whereas at higher concentrations both tryptophan and tyrosine participated in the interaction. Synchronous fluorescence spectra revealed that the microenvironment of tryptophan in HSA turned more hydrophilic upon exposure to GNP. The α-helical content of albumin was unaltered by GNP. Approximately 37 and 23% reduction in specific activity of HSA and BSA was observed due to GNP binding. In presence of warfarin and ibuprofen the binding constants of albumin–GNP complexes were altered. A very interesting observation not reported so far is the retained antioxidant activity of albumin in presence of GNP i.e. we believe that GNPs did not bind to the free sulfhydryl groups of albumin. However enhanced levels of copper binding were observed. We have also highlighted the differential response in albumin due to gold and silver nanoparticles which could be attributed to differences in the charge of the nanoparticle.     

A Stereological Approach for Estimation of Cellular Immunogold Labeling and Its Spatial Distribution in Oriented Sections Using the Rotator

Particulate gold labeling applied to ultrathin sections is a powerful approach for locating cellular proteins and lipids on thin sections of cellular structures and compartments. Effective quantitative methods now allow estimation of both density and distribution of gold labeling across aggregate organelles or compartment profiles. However, current methods generally use random sections of cells and tissues, and these do not readily present the information needed for spatial mapping of cellular quantities of gold label. Yet spatial mapping of gold particle labeling becomes important when cells are polarized or show internal organization or spatial shifts in protein/lipid localization. Here we have applied a stereological approach called the rotator to estimate cellular gold label and proportions of labeling over cellular compartments at specific locations related to a chosen cell axis or chosen cellular structures. This method could be used in cell biology for mapping cell components in studies of protein translocation, cell polarity, cell cycle stages, or component cell types in tissues. (J Histochem Cytochem 57:709–719, 2009)     

A novel biosensor for bovine serum albumin based on fluorescent self‐assembled sandwich bilayers

Fluorescent dyes butyl rhodamine B were assembled via a dl ‐cystenine intermediate onto quartz wafers whose surface had first adsorbed gold nanoparticles. Hence self‐assembled sandwich bilayers with nanocomposite structure were constructed which can be used as a biosensor for bovine serum albumin. The biosensor‐based self‐assembled monolayers (SAMs) are regenerable and have high sensitivity, five orders of magnitude higher than that of bulk solution phase sensing. The effects of existing forms of dyes on the fluorescence spectra of bilayers in the presence of bovine serum albumin were investigated. Copyright © 2008 John Wiley & Sons, Ltd.     

A highly sensitive resonance Rayleigh scattering and colorimetric assay for the recognition of propranolol in β‐adrenergic blocker

In this work, a highly sensitive, citrate anion‐capped gold nanoparticles (AuNPs)‐based assay for the determination of propranolol in real samples with resonance Rayleigh scattering (RRS) and colorimetry was developed. When AuNPs were prepared by the sodium citrate reduction method, citrate anions self‐assembled on the surface of AuNPs to form supramolecular complex anions. In BR 4.6 buffer solution, propranolol was positively charged and could bind with AuNPs to form larger aggregates through electrostatic force and hydrophobic effects. This results in remarkable enhancement of the RRS intensity and a color change in the AuNPs solution from red to blue via purple. Thus, a highly sensitive RRS and colorimetric assay the for detection of propranolol was developed with a linear range of 0.2–5.2 and 8–112 ng/ml, respectively. In addition, no difference was seen when comparing R‐propranolol with S‐propranolol, therefore, this method could not be used in the recognition of chiral propranolol. However, upon addition of other β‐adrenergic blockers, no phenomenon like that seen with propranolol was observed, meaning that this method can be used for determining the presence of propranolol in a mixture β‐adrenergic blockers. Finally, the optimum conditions, factors influencing the reaction, its mechanism and the reasons for enhancement of the RRS were discussed.     

An acetylcholinesterase biosensor based on graphene–gold nanocomposite and calcined layered double hydroxide

In this study, a novel acetylcholinesterase-based biosensor was fabricated. Acetylcholinesterase (AChE) was immobilized onto a glassy carbon electrode (GCE) with the aid of Cu–Mg–Al calcined layered double hydroxide (CLDH). CLDH can provide a bigger effective surface area for AChE loading, which could improve the precision and stability of AChE biosensor. However, the poor electroconductibility of CLDHs could lead to the low sensitivity of AChE biosensor. In order to effectively compensate the disadvantages of CLDHs, graphene–gold nanocomposites were used for improving the electron transfer rate. Thus, the graphene–gold nanocomposite (GN-AuNPs) was firstly modified onto the GCE, and then the prepared CLDH-AChE composite was immobilized onto the modified GCE to construct a sensitive AChE biosensor for pesticides detection. Relevant parameters were studied in detail and optimized, including the pH of the acetylthiocholine chloride (ATCl) solution, the amount of AChE immobilized on the biosensor and the inhibition time governing the analytical performance of the biosensor. The biosensor detected chlorpyrifos at concentrations ranging from 0.05 to 150 μg/L. The detection limit for chlorpyrifos was 0.05 μg/L.     

双单抗的免疫层析一步法用于早妊诊断的研究

在试管式、微孔式和斑点式的酶免测定法测定人绒毛膜促性腺激素（ＨＣＧ）的基础上,发展了应用双单克隆抗体的免疫层析一步法测定ＨＣＧ。此法用胶体金标记抗βＨＣＧ单克隆抗体,将抗αＨＣＧ单克隆抗体包被在硝酸纤维素膜上。无需分离步骤,特别是在进行测定时除加入样品外无需再加任何试剂,此方法特别迅速、简便,２～５ｍｉｎ即可得结果。凡ＨＣＧ浓度＞２５ＩＵ／Ｌ的样品可得到阳性结果。在人体血或尿中可能出现的高浓度的干扰物质,如抗坏血酸、乙酰水杨酸、雌二醇、蛋白质、胆红素、甘油三酯等对本测定均无干扰作用,在促黄体激素（ＬＨ）浓度高达５００ＩＵ／Ｌ时仍与ＨＣＧ没有交叉反应。能进行测定的最高值大于３００ＩＵ／ｍｌ,这表示,当ＨＣＧ浓度达到妊娠期的最高值时仍不会有假阴性结果。