Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry

Background

In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells.

Method

Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry.

Results

The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10⁵ and (0.85 ± 0.11) × 10⁵, respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC.

Conclusion

Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-11-43) contains supplementary material, which is available to authorized users.     

Irreversible enzyme-shuttle immunoassay

The concept of a competitive enzyme immunoassay that utilizes simultaneously the bound and the free analyte-enzyme conjugate (heterobifunctional conjugate) for signal generation in response to varying analyte concentrations in samples has been investigated. Two antigenic sites of the heterobifunctional conjugate are used in the assay for binding to immunoglobulins: the analyte derivative binds to an immobilized antibody, Ab(1), and the enzyme component binds to a spatially separated immobilized antibody, Ab(2). The analytical system is set up such that in the absence of analyte, the conjugate is predominantly bound in the compartment that contains Ab(1). With increasing concentration of native analyte in samples, an increasing concentration of native analyte in samples, an increasing amount of conjugate migrates to the second compartment that contains Ab(2). The enzyme bound in each compartment is used for signal generation. Mathematical models have been developed to determine the optimal conditions and to predict the performance of such dual-antibody systems. The theoretical predictions are supported by experimental results. The dual-antibody system has been compared with a conventional competitive enzyme immunoassay using the same reagents.     

Acoustic detection of stem and root borer Neoplocaederus ferrugineus (Coleoptera: Cerambycidae) in cashew

Stem and root borer Neoplocaederus ferrugineus (Linnaeus) (Coleoptera: Cerambycidae) is a severe pest of cashew (Anacardium occidentale L), which affects the cashew tree heavily. The larvae are tissue borers that remain concealed inside the tree and are very difficult to detect at the initial stage of infestation. Adult attractants are not identified, and manual checking of each tree in a plantation is not feasible and laborious. Acoustic technologies have been developed for the non-invasive and early detection of wood borers. The present study was conducted to characterize the acoustic sounds of various larval instars, tissues and field evaluation of the commercially available acoustic device. Burst analysis of different larval instars in cashew logs shows that there are no significant differences between the number of bursts and various larval instars. A relatively less number of bursts are found when the larvae are in the sapwood region than the cambium region. The empty cashew log and healthy trees do not produce any significant noise. The number of burst counts and impulses are used to predict the likelihood of infestation. The detection performance of the acoustic device under field conditions shows that infested trees are correctly detected with 91% accuracy. The possibility of not detecting healthy trees is 85%. This is the first report on the acoustic behaviour of cashew stem and root borer. The results of the study will help in better understanding the acoustic behaviour of the hidden pest and also be useful in the timely identification of stem and root borer-affected trees in cashew plantations.     

High-fat diet-induced lipidome perturbations in the cortex,hippocampus, hypothalamus,and olfactory bulb of mice

Given their important role in neuronal function, there has been an increasing focus on altered lipid levels in brain disorders. The effect of a high-fat (HF) diet on the lipid profiles of the cortex, hippocampus, hypothalamus, and olfactory bulb of the mouse brain was investigated using nanoflow ultrahigh pressure liquid chromatography-electrospray ionization-tandem mass spectrometry in the current study. For 8?weeks, two groups of 5-week-old mice were fed either an HF or normal diet (6 mice from each group analyzed as the F and N groups, respectively). The remaining mice in both groups then received a 4-week normal diet. Each group was then subdivided into two groups for another 4-week HF or normal diet. Quantitative analysis of 270 of the 359 lipids identified from brain tissue revealed that an HF diet significantly affected the brain lipidome in all brain regions that were analyzed. The HF diet significantly increased diacylglycerols, which play a role in insulin resistance in all regions that were analyzed. Although the HF diet increased most lipid species, the majority of phosphatidylserine species were decreased, while lysophosphatidylserine species, with the same acyl chain, were substantially increased. This result can be attributed to increased oxidative stress due to the HF diet. Further, weight-cycling (yo-yo effect) was found more critical for the perturbation of brain lipid profiles than weight gain without a preliminary experience of an HF diet. The present study reveals systematic alterations in brain lipid levels upon HF diet analyzed either by lipid class and molecular levels.     

Phytoremediation in Wetland Ecosystems: Progress,Problems, and Potential

Assessing the phytoremediation potential of wetlands is complex due to variable conditions of hydrology, soil/sediment types, plant species diversity, growing season, and water chemistry. Conclusions about long-term phytoremediation potential are further complicated by the process of ecological succession in wetlands. This review of wetlands phytoremediation addresses the role of wetland plants in reducing contaminant loads in water and sediments, including metals; volatile organic compounds (VOC), pesticides, and other organohalogens; TNT and other explosives; and petroleum hydrocarbons and additives. The review focuses on natural wetland conditions and does not attempt to review constructed wetland technologies. Physico-chemical properties of wetlands provide many positive attributes for remediating contaminants. The expansive rhizosphere of wetland herbaceous shrub and tree species provides an enriched culture zone for microbes involved in degradation. Redox conditions in most wetland soil/sediment zones enhance degradation pathways requiring reducing conditions. However, heterogeneity complicates generalizations within and between systems. Wetland phytoremediation studies have mainly involved laboratory microcosm and mesocosm technologies, with the exception of planted poplar communities. Fewer large-scale field studies have addressed remediation actions by natural wetland communities. Laboratory findings are encouraging with regards to phytoextraction and degradation by rhizosphere and plant tissue enzymes. However, the next phase in advancing the acceptance of phytoremediation as a regulatory alternative must demonstrate sustained contaminant removal by intact natural wetland ecosystems.     

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.     

On-line monitoring of enzymes in downstream processing by flow injection analysis (FIA)

Flow injection analysis (FIA) has been employed to automate enzyme assays for formate dehydrogenase (FDH) and l-leucine dehydrogenase (l-LeuDH). Coupled to a special sampling device the FIA assays were used to monitor on-line downstream processes, e.g. disintegration of microbial cells and cross-flow filtration of cell homogenates.     

Seasonal variation in hydrochloric acid, malic acid, and calciumions secreted by the trichomes of chickpea (Cicer arietinum)

A novel experimental set-up and method of recording of electrical potential differences in plants have been developed which enable continuous, 8-channel monitoring of electrical activity over extended periods of time using inserted, extracellular electrodes. The investigations were carried out on 21- to 23-day-old Helianthus annuus plants, and spontaneously-generated action potentials were recorded during monitoring sessions lasting for 3 days and nights. Characteristics of these spontaneous action potentials were elaborated, adopting as parameters their typical form, amplitude, duration, velocity, direction, and distance of propagation and frequency of occurrence in morphologically different parts of the plant, Variability, similarities, and interdependence of the above parameters in individual plants and in a group of 15 plants were determined. A hypothesis concerning propagation of action potentials in plants along specific impulse-propagating 'columns' is discussed. The frequency of generated impulses is highest at night and lowest in the day and also displays an apparent 24-h rhythm. Presumably this mechanism is under both endogenous and exogenous control and may be partly dependent on a biological clock.     

Modeling baseline conditions of ecological indicators: Marine renewable energy environmental monitoring

Ecological indicators are often collected to detect and monitor environmental change. Statistical models are used to estimate natural variability, pre-existing trends, and environmental predictors of baseline indicator conditions. Establishing standard models for baseline characterization is critical to the effective design and implementation of environmental monitoring programs. An anthropogenic activity that requires monitoring is the development of Marine Renewable Energy sites. Currently, there are no standards for the analysis of environmental monitoring data for these development sites. Marine Renewable Energy monitoring data are used as a case study to develop and apply a model evaluation to establish best practices for characterizing baseline ecological indicator data. We examined a range of models, including six generalized regression models, four time series models, and three nonparametric models. Because monitoring data are not always normally distributed, we evaluated model ability to characterize normal and non-normal data using hydroacoustic metrics that serve as proxies for ecological indicator data. The nonparametric support vector regression and random forest models, and parametric state-space time series models generally were the most accurate in interpolating the normal metric data. Support vector regression and state-space models best interpolated the non-normally distributed data. If parametric results are preferred, then state-space models are the most robust for baseline characterization. Evaluation of a wide range of models provides a comprehensive characterization of the case study data, and highlights advantages of models rarely used in Marine Renewable Energy environmental monitoring. Our model findings are relevant for any ecological indicator data with similar properties, and the evaluation approach is applicable to any monitoring program.     

Elevated Concentrations of Pesticides and PCBs in Soils at the Southern Caspian Sea (Iran) are Related to Land Use

Soil contamination by organochlorine pesticides or PCBs is almost undocumented for Iran. Here we report a soil survey in Mazandaran and Guilan provinces that hold >30% of the agricultural areas of Iran where pesticide use is widespread. Concentration of DDTs, HCHs, cyclodienes, and PCBs were measured in 45 soil samples from different agricultural land uses and forest land. The average concentrations of ∑DDT (37 μg kg^?1) and ∑HCH (21 μg kg^?1) in agricultural soils are among the largest ever reported and exceed international soil screening standards. All residues were larger in agricultural than in forest soil. Within agricultural land, ∑DDT were largest for tea gardens, lindane was largest in rice fields, and cyclodiens largest in citrus orchards. The ratio of (DDD + DDE)/DDT is an index of the extent of DDT degradation in soil and was lower in tea gardens than in other soils (0.7 versus 2–5), indicating either ongoing DDT input or lower degradation rate in the tea gardens that are more acid than the other soils (pH 4.5 versus 6.5–7.0). The o,p′–DDT/p,p′–DDT ratio was about 3 in forest soils, suggesting that DDT is derived from dicofol application and not from technical DDT as in agricultural soils. The PCB 28, 180, and 138 showed the highest mean concentration compared with other PCB congeners in all land uses. This survey is the first of this kind for Iran and illustrates that concentrations of organochlorine pesticide in soil are relatively large.