Therapeutic antibodies: Discovery and development using the ProteOn XPR36 biosensor interaction array system

Therapeutic monoclonal antibodies are becoming a significant and rapidly growing class of therapeutic pharmaceuticals. Their discovery and development requires fast and high-throughput methodologies for screening and selecting appropriate candidate antibodies having high affinity for the target as well as high specificity and low cross-reactivity. This study demonstrates the use of the ProteOn XPR36 protein interaction array system and its novel approach, termed One-Shot Kinetics, for the rapid screening and selection of high-affinity antibodies. This approach allows multiple quantitative protein binding analyses in parallel, providing association, dissociation, and affinity constants for several antibodies or supernatants simultaneously in one experiment. We show that the ProteOn XPR36 system is a valuable tool for use across multiple stages of the therapeutic antibody discovery and development process, enabling efficient and rapid screening after panning, affinity maturation, assay validation, and clone selection.     

Localization of two enzymes of the tetrahydrobiopterin biosynthetic pathway in embryonic chick retina.

Tetrahydrobiopterin (BH4) is an essential co-factor for the biosynthesis of catecholamine-type neurotransmitters and of nitric oxide (NO). The expression of the enzymes catalyzing the first two steps of the BH4 biosynthetic pathway was studied in the developing chicken retina by in situ hybridization and immunocytochemistry. GTP-cyclohydrolase-I (GTP-CH-I) and 6-pyruvoyl-tetrahydropterin synthase (PTPS) were already expressed in the undifferentiated and proliferating retina of E7. At stage E11 both enzymes were expressed in photoreceptors, amacrine cells, displaced amacrine cells, and ganglion cells, and in the plexiform layers in which synaptic connections take place. At stage E18 the labeling was comparable to E11 but appeared to be more concentrated in photoreceptors and ganglion cells.     

Synergistic effects of abiotic stresses in plants: a case study of nitrogen limitation and saturating light intensity in Arabidopsis thaliana

Under natural conditions, plants are regularly exposed to combinations of stress factors. A common example is the conjunction between nitrogen (N) deficiency and excess light. The combined effect of stress factors is often ignored in studies using controlled conditions, possibly resulting in misleading conclusions. To address this issue, the present study examined the physiological behavior of Arabidopsis thaliana under the effect of varying nitrogen levels and light intensities. The joint influence of low N and excess light had an adverse effect on plant growth, chlorophyll and anthocyanin concentrations, photochemical capacity and the abundance of proteins involved in carbon assimilation and antioxidative metabolism. In contrast, no adverse physiological responses were observed for plants under either nitrogen limitation or high light (HL) intensity conditions (i.e. single stress). The underlying mechanisms for the increased growth in conditions of HL and sufficient nitrogen were a combination of chlorophyll accumulation and an increased number of proteins involved in C3 carbon assimilation, amino acids biosynthesis and chloroplast development. In contrast, combined stress conditions shifts plants from growth to survival by displaying anthocyanin accumulation and an increased number of proteins involved in catabolism of lipids and amino acids as energy substrates. Ultimately switching plants development from growth to survival. Our results suggest that an assessment of the physiological response to the combined effect of multiple stresses cannot be directly extrapolated from the physiological response to a single stress. Specifically, the synergistic interaction between N deficiency and saturating light in Arabidopsis plants could not have been modeled via only one of the stress factors.     

A Simple Method for Assessment of Human Anti-Neu5Gc Antibodies Applied to Kawasaki Disease

N-Glycolylneuraminic acid (Neu5Gc) is an immunogenic sugar of dietary origin that metabolically incorporates into diverse native glycoconjugates in humans. Anti-Neu5Gc antibodies are detected in all human sera, though with variable levels and epitope-recognition profiles. These antibodies likely play a role in several inflammation-mediated pathologies including cardiovascular diseases and cancer. In cancer, they have dualistic and opposing roles, either stimulating or repressing disease, as a function of their dose, and some of these antibodies serve as carcinoma biomarkers. Thus, anti-Neu5Gc antibodies may signify risk of inflammation-mediated diseases, and changes in their levels could potentially be used to monitor disease progression and/or response to therapy. Currently, it is difficult to determine levels of anti-Neu5Gc antibodies in individual human samples because these antibodies recognize multiple Neu5Gc-epitopes. Here we describe a simple and specific method for detection and overall estimation of human anti-Neu5Gc antibodies. We exploit the difference between two mouse models that differ only by Neu5Gc-presence (wild-type) or Neu5Gc-absence (Cmah^−/− knockout). We characterize mouse serum from both strains by HPLC, lectin and mass-spectrometry analysis and show the target Neu5Gc-epitopes. We then use Cmah^−/− knockout sera to inhibit all non-Neu5Gc-reactivity followed by binding to wild-type sera to detect overall anti-Neu5Gc response in a single assay. We applied this methodology to characterize and quantify anti-Neu5Gc IgG and IgA in sera of patients with Kawasaki disease (KD) at various stages compared to controls. KD is an acute childhood febrile disease characterized by inflammation of coronary arteries that untreated may lead to coronary artery aneurysms with risk of thrombosis and myocardial infarction. This estimated response is comparable to the average of detailed anti-Neu5Gc IgG profile analyzed by a sialoglycan microarray. Both assays revealed an elevated response in acute KD patients with normal coronaries compared to patients with aneurysm or dilated coronaries. Implications of these findings are discussed.     

The Impact of a 24 Month Housing First Intervention on Participants’ Body Mass Index and Waist Circumference: Results from the At Home / Chez Soi Toronto Site Randomized Controlled Trial

Research suggests that individuals experiencing homelessness have high rates of overweight and obesity. Unhealthy weights and homelessness are both associated with increased risk of poor health and mortality. Using longitudinal data from 575 participants at the Toronto site of the At Home/Chez Soi randomized controlled trial, we investigate the impact of receiving a Housing First intervention on the Body Mass Index (BMI) and waist circumference of participants with moderate and high needs for mental health support services. The ANCOVA results indicate that the intervention resulted in no significant change in BMI or waist circumference from baseline to 24 months. The findings suggest a need for a better understanding of factors contributing to overweight, obesity, and high waist circumference in populations who have histories of housing precarity and experience low-income in tandem with other concerns such as mental illness and addictions.

Trial Registration

International Standard Randomized Control Trial Number Register ISRCTN42520374     

Metabolism of lignin related aromatic compounds by Aspergillus japonicus

Archives of Microbiology - Aspergillus japonicus metabolizes a wide variety of aromatic compounds, some of them resulting from lignin degradation. The efficient conversion of such compounds is...     

Prediction of response to robot-aided motor neuro-rehabilitation of children with cerebral palsy

Robot-aided neuro-rehabilitation is increasingly being incorporated into rehabilitation practices. The aim of this study was to search for patterns in the data acquired by a robot in the baseline evaluation session which could predict progress over the next therapy sessions. Kinematic and kinetic data were acquired during robot-mediated evaluation sessions of 12 children with motor impairments due to hemiplegic cerebral palsy (CP). Time and wavelets features were extracted from the data and used for K-means clustering. The data were labeled by the Quality of Upper Extremity Skills Test (QUEST) and the gradient of improvement change in the QUEST between the baseline assessment and therapy follow-up one month after completion of 16 robot-mediated therapy sessions. Two distinct clusters segregated these 12 children into performers and non-performers in terms of the QUEST.