An integrative network analysis framework for identifying molecular functions in complex disorders examining major depressive disorder as a test case

In addition to the psychological depressive phenotype, major depressive disorder (MDD) patients are also associated with underlying immune dysregulation that correlates with metabolic syndrome prevalent in depressive patients. A robust integrative analysis of biological pathways underlying the dysregulated neural connectivity and systemic inflammatory response will provide implications in the development of effective strategies for the diagnosis, management and the alleviation of associated comorbidities. In the current study, focusing on MDD, we explored an integrative network analysis methodology to analyze transcriptomic data combined with the meta-analysis of biomarker data available throughout public databases and published scientific peer-reviewed articles. Detailed gene set enrichment analysis and complex protein–protein, gene regulatory and biochemical pathway analysis has been undertaken to identify the functional significance and potential biomarker utility of differentially regulated genes, proteins and metabolite markers. This integrative analysis method provides insights into the molecular mechanisms along with key glycosylation dysregulation underlying altered neutrophil-platelet activation and dysregulated neuronal survival maintenance and synaptic functioning. Highlighting the significant gap that exists in the current literature, the network analysis framework proposed reduces the impact of data gaps and permits the identification of key molecular signatures underlying complex disorders with multiple etiologies such as within MDD and presents multiple treatment options to address their molecular dysfunctio

Vulnerability to stress: personality facet of vulnerability is associated with cardiovascular adaptation to recurring stress

It is increasingly suggested that personality traits are critical to understanding patterns of cardiovascular stress adaptation. However, studies have focused on higher-order traits with no research having examined underlying facet effects to repeated stress. The examination of facets provides a more granular examination, which has the potential to identify specific personality components that are relevant within the context of psychophysiological stress adaptation. This study objective was to determine if the underlying facets which encapsulate the dimension of emotional stability, are associated with cardiovascular adaptation to recurring stress. Continuous cardiovascular monitoring and psychometric measures were collated from 79 healthy young male and female adults, across a protocol of recurring active stress tasks. Multiple regression analysis revealed that the facet of vulnerability was associated with systolic and diastolic blood pressure adaptation across the protocol. More specifically, vulnerability was negatively associated with adaptation to recurring stress, such that those highest in vulnerability displayed a sensitization to the recurring stressor. No significant effects emerged for any other facet. Importantly, this research adds to the existing literature examining stress adaptation and has implications for future research on the relevance of examining facet effects. This study is the first to implicate the personality facet of vulnerability which encapsulates an individual's tendency to feel unable to cope with stress and becoming hopeless when faced with emergency situations, in the context of cardiovascular stress adaptation. Taken together, this study suggests that the facet of vulnerability is a critical component to consider in the context of cardiovascular stress adaptation

Development of a Convenient Competitive ELISA for the Detection of the Free and Protein-Bound Nonhuman Galactosyl-α-(1,3)-Galactose Epitope Based on Highly Specific Chicken Single-Chain Antibody Variable-Region Fragments

The presence of the nonhuman galactosyl-α-(1,3)-galactose (Gal-α-(1,3)-Gal) carbohydrate epitope on a number of recombinant therapeutic proteins has recently been reported, renewing interest in this immunogenic carbohydrate epitope. It is well-known that this motif is the primary contributing factor in hyperacute rejection of porcine organ xenograft, due to the existence of natural antibodies against this epitope in human serum. Though the number of epitopes on recombinant glycoproteins may be low when compared directly to whole tissue, circulating anti-Gal-α-R immunoglobulins can still induce anaphylaxis. Therefore, there is a need for rapid and convenient methods for detection and monitoring of this epitope in biopharmaceuticals produced in recombinant mammalian systems. To this end, we have generated immune-challenged chicken single-chain antibody variable-region fragment (scFv) libraries targeting the Gal-α-(1,3)-Gal motif and have selected a panel of scFv's that bind the target. We have used one of these antibodies to develop a competitive ELISA for both free and protein-bound Gal-α-(1,3)-Gal and have demonstrated that the ELISA is specific for the target and can be used to determine the loading of the target on glycoproteins. This competitive ELISA will provide a convenient method of detecting and quantifying Gal-α-(1,3)-Gal on therapeutic glycoproteins

Effect of Nanoparticle Stabilization and Physicochemical Properties on Exposure Outcome: Acute Toxicity of Silver Nanoparticle Preparations in Zebrafish (<i>Danio rerio</i>)

Nanotechnology has vast potential for expanded development and novel application in numerous sectors of society. With growing use and applications, substantial production volumes and associated environmental release can be anticipated. Exposure effect of nanoparticles (NP) on biological systems may be intrinsic to their physicochemical properties introducing unknown associated risk. Herein, we expand the knowledge of health and environmental impact of silver nanoparticles (AgNPs), testing the acute toxicity of 14 AgNP preparations on developing zebrafish embryos (<i>Danio rerio</i>). Toxicological end points, including mortality, hatching rate, and heart rate were recorded. Concentration, stabilization agent and physicochemical properties were monitored as contributing outcome factors. Our findings indicate wide ranging LC₅₀ 24 h postfertilization values (0.487 ppm (0.315, 0.744 95% CI) to 47.89 ppm (18.45, 203.49 95% CI)), and indicate surface charge and ionic dissolution as key contributory factors in AgNP exposure outcome

Construction of a Natural Mucin Microarray and Interrogation for Biologically Relevant Glyco-Epitopes

Mucins are the principal components of mucus, and mucin glycosylation has important roles in defense, microbial adhesion, immunomodulation, inflammation, and cancer. Mucin expression and glycosylation are dynamic, responding to changes in local environment and disease. Potentially hundreds of heterogeneous glycans can substitute one mucin molecule, and it is difficult to identify biologically accessible glyco-epitopes. Thirty-seven mucins, from the reproductive and gastrointestinal (GI) tracts of six species (bovine, ovine, equine, porcine, chicken, and deer) and from two human-derived cell lines, were purified. Following optimization of mucin printing and construction of a novel mucin microarray, the glycoprofiles of the whole mucins on the microarray were compared using a panel of lectins and one antibody. Accessible glyco-motifs of GI mucins varied according to species and localization of mucin origin, with terminal fucose, the sialyl T-antigen, and <i>N</i>-linked oligosaccharides identified as potentially important. The occurrence of T- and sialyl T-antigen varied in bovine and ovine reproductive tract mucins, and terminal <i>N</i>-acetylgalactosamine (GalNAc) and sulfated carbohydrates were detected. This study introduces natural mucin microarrays as an effective tool for profiling mucin glyco-epitopes and highlights their potential for discovery of biologically important motifs in bacterial–host interactions and fertility

Schematic of lectin microarray profiling process.

<p>Schematic of lectin microarray profiling process.</p

Unsupervised hierarchical clustering of lectin microarray profiles for intact urine uEVs and purified THP from three healthy individuals.

<p>LM data for all uEV (donors D1, D2, and D3) and THP samples (donors D1, D2 and D4) normalized by rescaling (range 0 to 65200). Individual technical replicates are indicated by designation of 1 to 5. Two groups are evident at 0.28 similarity, three groups at 0.57 similarity. Clustering based on Euclidean distance, complete linkage method. Responses and clustering shown are typical of those obtained from a minimum of 10 healthy uEV enrichment and profiling experiments.</p

Competitive inhibition of uEV interactions with lectin microarray using six different sugars.

<p>Data normalized to the response of LEL to show the relative inhibition through competition with 50(<b>A</b>) Lac, (<b>B</b>) Gal, (<b>C</b>) Man, (<b>D</b>) GalNAc, and (<b>E</b>) and Fuc (<b>F</b>). To evaluate 50 mM GlcNAc inhibition (<b>F</b>), data was normalized to the response of RCA-I. Mean data is representative of inhibition for a single biological sample experiment conducted with 3 technical replicates. Error bars represent ± average deviation. Significance (** = <i>p</i>≤0.01, * = <i>p</i>≤0.05) determined by two-tailed, two-sampled unequal variance Student’s t-test. Arrows mark reductions in intensity greater than 20% with <i>p</i>>0.05.</p

Down-regulated genes common to all treatments.

<p>3′SL –3′sialyllactose; 6′SL –6′sialyllactose; 3′+6′SL – combined treatment of 3′- and 6′-sialyllactose; FC – fold change; pval – p-value.</p

Surface Glycosylation Profiles of Urine Extracellular Vesicles

<div><p>Urinary extracellular vesicles (uEVs) are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications.</p></div