Complementary Effects of Carbamylated and Citrullinated LL37 in Autoimmunity and Inflammation in Systemic Lupus Erythematosus

LL37 acts as T-cell/B-cell autoantigen in Systemic lupus erythematosus (SLE) and psoriatic disease. Moreover, when bound to “self” nucleic acids, LL37 acts as “danger signal,” leading to type I interferon (IFN-I)/pro-inflammatory factors production. T-cell epitopes derived from citrullinated-LL37 act as better antigens than unmodified LL37 epitopes in SLE, at least in selected HLA-backgrounds, included the SLE-associated HLA-DRB1*1501/HLA-DRB5*0101 backgrounds. Remarkably, while “fully-citrullinated” LL37 acts as better T-cell-stimulator, it loses DNA-binding ability and the associated “adjuvant-like” properties. Since LL37 undergoes a further irreversible post-translational modification, carbamylation and antibodies to carbamylated self-proteins other than LL37 are present in SLE, here we addressed the involvement of carbamylated-LL37 in autoimmunity and inflammation in SLE. We detected carbamylated-LL37 in SLE-affected tissues. Most importantly, carbamylated-LL37-specific antibodies and CD4 T-cells circulate in SLE and both correlate with disease activity. In contrast to “fully citrullinated-LL37,” “fully carbamylated-LL37” maintains both innate and adaptive immune-cells’ stimulatory abilities: in complex with DNA, carbamylated-LL37 stimulates plasmacytoid dendritic cell IFN-α production and B-cell maturation into plasma cells. Thus, we report a further example of how different post-translational modifications of a self-antigen exert complementary effects that sustain autoimmunity and inflammation, respectively. These data also show that T/B-cell responses to carbamylated-LL37 represent novel SLE disease biomarkers.     

A Novel Cartesian Plot Analysis for Fixed Monolayers That Relates Cell Phenotype to Transfer of Contents between Fibroblasts and Cancer Cells by Cell-Projection Pumping

We recently described cell-projection pumping as a mechanism transferring cytoplasm between cells. The uptake of fibroblast cytoplasm by co-cultured SAOS-2 osteosarcoma cells changes SAOS-2 morphology and increases cell migration and proliferation, as seen by single-cell tracking and in FACS separated SAOS-2 from co-cultures. Morphological changes in SAOS-2 seen by single cell tracking are consistent with previous observations in fixed monolayers of SAOS-2 co-cultures. Notably, earlier studies with fixed co-cultures were limited by the absence of a quantitative method for identifying sub-populations of co-cultured cells, or for quantitating transfer relative to control populations of SAOS-2 or fibroblasts cultured alone. We now overcome that limitation by a novel Cartesian plot analysis that identifies individual co-cultured cells as belonging to one of five distinct cell populations, and also gives numerical measure of similarity to control cell populations. We verified the utility of the method by first confirming the previously established relationship between SAOS-2 morphology and uptake of fibroblast contents, and also demonstrated similar effects in other cancer cell lines including from melanomas, and cancers of the ovary and colon. The method was extended to examine global DNA methylation, and while there was no clear effect on SAOS-2 DNA methylation, co-cultured fibroblasts had greatly reduced DNA methylation, similar to cancer associated fibroblasts.     

Changes in bioactive compounds and antioxidant activity during convective drying of murta (Ugni molinae T.) berries

Murta (Ugni molinae T.) berries were air‐dried at five temperatures (40, 50, 60, 70 and 80 °C), and the changes in β‐carotene, phenolic acids, total phenolic and flavonoid contents and antioxidant capacities (DPPH and ORAC) were investigated. The berries showed a high content of β‐carotene, which decreased during drying temperature between 40 °C and 80 °C. Free and bound phenolic acids were also determined, showing gallic acid to be the prevalent phenolic acid. Total phenolic and flavonoid contents in the dried berries showed a higher decrease at lower temperature due to longer drying time. The radical‐scavenging activity also showed higher antioxidant activity at higher drying temperatures (70–80 °C) than at lower drying temperatures (40–50 °C). Total phenolic content (TPC) and flavonoids showed good correlation with antioxidant capacity. Murta berries proved to be an excellent source of antioxidants and bioactive compounds and are therefore a potential ingredient for new functional food products.     

Development of a questionnaire to assay recalled liking for salt,sweet and fat

Liking for a sweet, salty or fatty diet may induce overconsumption of simple carbohydrates, sodium or lipids. Measuring overall liking of the corresponding sensory sensations contributes to understanding the determinants of dietary behaviours. However, no standardized validated questionnaire assaying these sensations is currently available. In the present study, we developed a web-based questionnaire, “PrefQuest”, which measures recalled liking for the following four sensations: salt, fat and salt, sweet, fat and sweet. PrefQuest included four types of items: (1) liking for sweet, fatty-sweet and fatty-salty foods, (2) preferences in the level of seasoning by adding salt, sweeteners, or fat, (3) preferences for types of dishes in a restaurant menu and (4) overall questions about sweet-, salt- and fat-related behaviours. A development study (n = 198) demonstrated that PrefQuest was feasible, well-perceived, only lasted about 20 min and that items were repeatable (overall mean intra-class correlation coefficient: 0.77, SD 0.08). Construct validity was then evaluated on a larger population sample (n = 47 803). The underlying structure within each of the four sensations was determined by exploratory factor analysis and then internally validated by confirmatory factor analysis. After a selection of the most relevant items, the salt, fat-and-salt, sweet, and fat-and-sweet scales exhibited a theoretically meaningful factor structure, unidimensional for the salt scale and with interrelated sub-dimensions for the sweet, fat-and-salt, and fat-and-sweet scales. Based on the fat-and-sweet and fat-and-salt scales, a fat model was also unveiled. For each factor, internal consistency as well as convergent and divergent validities were demonstrated. Overall, PrefQuest is an internally valid and original tool that can be used to assay recalled liking for sweet, salt, and fat considered altogether or separately as fat and salt or fat and sweet, and can be applied to large population surveys.     

Identification of unbalance forces by metaheuristic search algorithms

This paper discusses the identification of parameters in rotary systems, namely, the unbalance magnitude, phase and position in the rotor system. These parameters can be identified using the measured orbits in the hydrodynamic bearings. The oil film forces are evaluated in the different positions of the orbit of the journal and are applied to the model of the shaft. The model, integrated in time domain, allows with an assumed unbalance, to simulate the orbits. The objective function is basically the difference between measured and simulated orbits, and its minimum corresponds to the identified unbalance amount, phase and position along the shaft. With respect to traditional model based identification procedures, this approach using oil film forces instead of oil film linearized stiffness and damping coefficients, and unfiltered orbits instead of 1X vibration components is suitable to deal with non-linear behaviour of the system.     

Kinetic study of a hen-egg white lysozyme-based oenological preparation

The catalytic activity of a lysozyme-based oenological preparation toward the specific substrates i) glycol chitosan, as a non-cellular synthetic substrate, and ii) Oenococcus oeni, as a microbial substrate for muramidase activity, was investigated in wine-like acidic medium (tartaric buffer, pH 3.2). The good reproducibility and reliability of results revealed that both substrates are useful for lysozyme kinetics studies. Both the chitinolytic and muramidase activity of lysozyme were affected by uncompetitive substrate inhibition. However, the O. oeni effect occurred at very high concentration with respect to the typical wine bacterial content, even when microbial population reaches its maximum growth level during malolactic fermentation. The optimum pH and temperature for lysozyme activity were very far from the usual wine handling conditions, but residual enzyme activity was observed at pH 3.2 and 20°C, confirming that the oenological application of lysozyme is a useful practice.