Analytical techniques for multiplex analysis of protein biomarkers

Introduction: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. Areas covered: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. Expert commentary: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine

Analytical techniques for multiplex analysis of protein biomarkers

Introduction: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. Areas covered: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. Expert commentary: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine.</div

Frequency, mutual exclusivity and clinical associations of myositis autoantibodies in a combined European cohort of idiopathic inflammatory myopathy patients

Objectives: To determine prevalence and co-existence of myositis specific autoantibodies (MSAs) and myositis associated autoantibodies (MAAs) and associated clinical characteristics in a large cohort of idiopathic inflammatory myopathy (IIM) patients. Methods: Adult patients with confirmed IIM recruited to the EuroMyositis registry (n = 1637) from four centres were investigated for the presence of MSAs/MAAs by radiolabelled-immunoprecipitation, with confirmation of anti-MDA5 and anti-NXP2 by ELISA. Clinical associations for each autoantibody were calculated for 1483 patients with a single or no known autoantibody by global linear regression modelling. Results: MSAs/MAAs were found in 61.5% of patients, with 84.7% of autoantibody positive patients having a sole specificity, and only three cases (0.2%) having more than one MSA. The most frequently detected autoantibody was anti-Jo-1 (18.7%), with a further 21 specificities each found in 0.2–7.9% of patients. Autoantibodies to Mi-2, SAE, TIF1, NXP2, MDA5, PMScl and the non-Jo-1 tRNA-synthetases were strongly associated (p < 0.001) with cutaneous involvement. Anti-TIF1 and anti-Mi-2 positive patients had an increased risk of malignancy (OR 4.67 and 2.50 respectively), and anti-SRP patients had a greater likelihood of cardiac involvement (OR 4.15). Interstitial lung disease was strongly associated with the anti-tRNA synthetases, antiMDA5, and anti-U1RNP/Sm. Overlap disease was strongly associated with anti-PMScl, anti-Ku, anti-U1RNP/Sm and anti-Ro60. Absence of MSA/MAA was negatively associated with extra-muscular manifestations. Conclusions: Myositis autoantibodies are present in the majority of patients with IIM and identify distinct clinical subsets. Furthermore, MSAs are nearly always mutually exclusive endorsing their credentials as valuable disease biomarkers

Zein Nanoparticles Uptake and Translocation in Hydroponically Grown Sugar Cane Plants

The main objective of this study was to investigate the uptake and translocation of positively charged zein nanoparticles (ZNPs) in hydroponically grown sugar cane plants. Fluorescent ZNPs (spherical and measuring an average diameter 135 ± 3 nm) were synthesized by emulsion-diffusion method from FITC-tagged zein. Fluorescent measurement following digestion of plant tissue indicated that sugar cane roots had a significant adhesion of ZNPs, 342.5 ± 24.2 μg NPs/mg of dry matter, while sugar cane leaves contained a very limited amount, 12.9 ± 1.2 μg NPs/mg dry matter for high dose(1.75 mg/ml) after 12 h. Confocal microscopy studies confirmed presence of fluorescent ZNPs in the epidermis and endodermis of the root system. Given their ability to adhere to roots for extended periods of time, ZNPs are proposed as effective delivery systems for agrochemicals to sugar cane plants, but more studies are needed to identify effect of nanoparticle exposure to health of the plant

Perspectives of Molecularly Imprinted Polymer-Based Drug Delivery Systems in Ocular Therapy

Although the human eye is an easily accessible sensory organ, it remains a challenge for drug administration due to the presence of several anatomical and physiological barriers which limit the access of drugs to its internal structures. Molecular imprinting technology may be considered the avant-garde approach in advanced drug delivery applications and, in particular, in ocular therapy. In fact, molecularly imprinted polymers hold the promise to compensate for the current shortcomings of the available arsenal of drug delivery systems intended for ocular therapy. The present manuscript aims to review the recent advances, the current challenges and most importantly to raise awareness on the underexplored potential and future perspectives of molecularly imprinted polymer-based drug delivery systems intended for the treatment of eye diseases

Toxicity and biouptake of lead and arsenic by Daphnia pulex

Cataracts are responsible for half of the world blindness, surgery being the only viable treatment. Lutein, a naturally occurring carotenoid in the eye, has the potential to reduce cataract progression by protecting the eye from photo-oxidative stress. To restore the eye\u27s natural line of defense against photo-oxidative stress, a formulation was developed using zein and poly(lactic-co-glycolic acid) nanoparticles (NPs) embedded in an optimized bioadhesive thermosensitive gel for the delivery of lutein via topical application. Cataracts were induced in Crl:WI rats via selenite injection at 13 days post-partum, followed by 7 days of treatment with free lutein or lutein-loaded NPs administered orally or topically. Cataract severity was significantly reduced in rats treated with topical applications of lutein-loaded NPs compared to the positive control, while no significant differences were observed in rats treated with other lutein formulations including oral and topically applied free lutein

Flow electrochemical analyses of zinc by stripping voltammetry on graphite felt electrode.

International audienceA flow sensor for trace analysis of zinc, using graphite felt as working electrode is reported here. A flow cell, well-adapted to 3-D porous electrodes and capable to do both the preconcentration step at a cathodic potential and the stripping of the zinc was successfully developed. It was demonstrated that this cell allows to obtain better electrochemical signals for Zn(2+) compared to a standard three-electrodes cell and that the percolation during accumulation increases the kinetics of electrodeposition. The influence on Zn(2+) signal of the deposition potential, the time of deposition and the flow rate was studied. The resulting sensor shows a linear response towards Zn(2+) with a linear range of 10(-6)-10(-4)M and a limit of detection of 5×10(-7) M for an analysis time of 5 min. The interferences study showed that the Cr(3+), Pb(2+), Cd(2+) ions have a small effect on the Zn electrochemical signal, whereas Fe(3+), Cu(2+), Co(2+) and Ni(2+) ions strongly influence it. The electrode was tested on real samples (tap water spiked with Zn(2+), food supplement) with a good recovery by applying the standard addition method

Zein Nanoparticles Uptake by Hydroponically Grown Soybean Plants

In the interest of developing and characterizing a polymeric nanoparticle pesticide delivery vehicle to soybeans, zein nanoparticle (ZNP) uptake by the roots and biodistribution to the leaves of soybean plants was measured. Zein was tagged with fluorescein isothiocyanate (FITC) and made into nanoparticles (135 ± 3 nm diameter. 0.202 ± 0.034 PDI and 81 ± 4 mV zeta-potential at pH 6) using an emulsion-diffusion method. After 10 days of hydroponic exposure, association between particles and roots of plants was found to vary based on bulk nanoparticle concentration. While 0.37 mg NP/mg dry weight were detected in roots immersed in 0.88 mg NP/mL nanoparticle suspension, 0.58 mg NP/mg dry weight associated with roots immersed in a high dose nanoparticle suspension of 1.75 mg NP/mL at 10 days. Nanoparticle root uptake followed second order kinetics. A small amount of increased fluorescence was detected in the hydroponically exposed plant\u27s leaves, suggesting that either small amounts of particles or other fluorescent contaminants of zein were up taken by the roots and biodistributed within the plant. To the authors\u27 knowledge, this is the first study in which the uptake and time-dependent association between polymeric nanoparticles and soybeans are quantified