Over the past two decades, library-based display technologies have been staggeringly optimized since their appearance in order to mimic the process of natural molecular evolution. Display technologies are essential for the isolation of specific high-affinity binding molecules (proteins, polypeptides, nucleic acids and others) for diagnostic and therapeutic applications in cancer, infectious diseases, autoimmune, neurodegenerative, inflammatory pathologies etc. Applications extend to other fields such as antibody and enzyme engineering, cell-free protein synthesis and the discovery of protein–protein interactions. Phage display technology is the most established of these methods but more recent fully in vitro alternatives, such as ribosome display, mRNA display, cis-activity based (CIS) display and covalent antibody display (CAD), as well as aptamer display and in vitro compartmentalization, offer advantages over phage in library size, speed and the display of unnatural amino acids and nucleotides. Altogether, they have produced several molecules currently approved or in diverse stages of clinical or preclinical testing and have provided researchers with tools to address some of the disadvantages of peptides and nucleotides such as their low affinity, low stability, high immunogenicity and difficulty to cross membranes. In this review we assess the fundamental technological features and point out some recent advances and applications of display technologies.

Bovine mastitis causes changes in the milk and serum proteomes. Here changes in both proteomes caused by naturally occurring subclinical and clinical mastitis have been characterised and quantified. Milk and serum samples from healthy dairy cows (n = 10) were compared to those of cows with subclinical (n = 12) and clinical mastitis (n = 10) using tandem mass tag (TMT) proteomics. Proteins that significantly increased or decreased in milk (n = 237) or serum (n = 117) were quantified and classified by the type of change in subclinical and clinical mastitis. A group of the proteins (n = 38) showed changes in both milk and serum a number of which decreased in the serum but increased in milk, suggesting a particular role in host defence for maintaining and restoring homeostasis during the disease. Proteins affected by bovine mastitis included proteins in host defence and coagulation pathways. Investigation of the modified proteomes in milk and serum was assessed by assays for haptoglobin, serum amyloid A and α1 acid glycoprotein validating the results obtained by quantitative proteomics. Alteration of abundance patterns of milk and serum proteins, together with pathway analysis reveal multiple interactions related to proteins affected by mastitis. Data are available via ProteomeXchange with identifier PXD022595. SIGNIFICANCE: Mastitis is the most serious condition to affect dairy cows and leads to reduced animal welfare as well as having a negative economic effect for the dairy industry. Proteomics has previously identified changes in abundance of milk proteins during mastitis, but there have been few investigations addressing changes that may affect proteins in the blood during the infection. In this study, changes in the abundance of proteins of milk and serum, caused by naturally occurring mastitis have been characterised by proteomics using a quantitative approach and both subclinical and clinical cases of mastitis have been investigated. In both milk and serum, change in individual proteins was determined and classified into varying types of altering abundance, such as increasing in subclinical mastitis, but showing no further increase in clinical mastitis. Of special interest were the proteins that altered in abundance in both milk and serum which either showed similar trends - increasing or decreasing in both biological fluids or showed reciprocal change decreasing in serum but increasing in milk. As well as characterising proteins as potential markers of mastitis and the severity of the disease, these results provide insight into the pathophysiology of the host response to bovine mastitis.

Increasing evidence implicates lysosomal dysfunction in the pathogenesis of neurodegenerative diseases, including the rare inherited lysosomal storage disorders (LSDs) and the most common neurodegenerative diseases, such as Alzheimer9s and Parkinson9s disease (AD and PD). Although the triggers of the lysosomal impairment may involve the accumulated macromolecules or dysfunction of the lysosomal enzymes, the role of the lysosomal glycocalyx in the lysosomal (dys)function has not been studied. The goal of this work was to analyze whether there are changes in the lysosomal glycocalyx in a cellular model of a LSD Niemann-Pick type C disease (NPC). Using the ferrofluid nanoparticles we isolated lysosomal organelles from NPC1-null and CHOwt cells. The magnetically isolated lysosomal fractions were enriched with the lysosomal marker protein LAMP1 and showed the key features of NPC disease: 3-fold higher cholesterol content and 4–5 fold enlarged size of the particles compared with the lysosomal fractions of wt cells. These lysosomal fractions were further processed to isolate lysosomal membrane proteins using Triton X-114 and their N-glycome was analyzed by HILIC-UPLC. N-glycans presented in each chromatographic peak were elucidated using MALDI-TOF/TOF-MS. We detected changes in the N-glycosylation pattern of the lysosomal glycocalyx of NPC1-null versus wt cells which involved high-mannose and sialylated N-glycans. To the best of our knowledge this study is the first to report N-glycome profiling of the lysosomal glycocalyx in NPC disease cellular model and the first to report the specific changes in the lysosomal glycocalyx in NPC1-null cells. We speculate that changes in the lysosomal glycocalyx may contribute to lysosomal (dys)function. Further glycome profiling of the lysosomal glycocalyx in other LSDs as well as the most common neurodegenerative diseases, such as AD and PD, is necessary to better understand the role of the lysosomal glycocalyx and to reveal its potential contribution in lysosomal dysfunction leading to neurodegeneration.