Validation of Reference Genes for the Relative Quantification of Gene Expression in Human Epicardial Adipose Tissue

BACKGROUND: Relative quantification is a commonly used method for assessing gene expression, however its accuracy and reliability is dependent upon the choice of an optimal endogenous control gene, and such choice cannot be made a priori. There is limited information available on suitable reference genes to be used for studies involving human epicardial adipose tissue. The objective of the current study was to evaluate and identify optimal reference genes for use in the relative quantification of gene expression in human epicardial fat depots of lean, overweight and obese subjects. METHODOLOGY/PRINCIPAL FINDINGS: Some of the commonly used reference genes including 18S, ACTB, RPL27, HPRT, CYCA, GAPDH, RPLPO, POLR2A and B2M were quantified using real-time PCR analysis. The expression stability of these genes was evaluated using Genorm, Normfinder and Bestkeeper algorithms. In addition, the effect of sample size on the validation process was studied by randomly categorizing subjects in two cohorts of n = 2 and n = 33. CONCLUSIONS/SIGNIFICANCE: CYCA, GAPDH and RPL27 were identified as the most stable genes common to all three algorithms and both sample sizes. Their use as reference gene pairs might contribute to the enhanced robustness of relative quantification in the studies involving the human epicardial adipose tissue

Active Whey Protein Edible Films and Coatings Incorporating Lactobacillus buchneri for Penicillium nordicum Control in Cheese

Fungal contamination of food is responsible for health issues and food waste. In this work, the incorporation of a lactic acid bacteria (LAB) with antifungal properties (Lactobacillus buchneri UTAD104) into whey protein-based films and coatings was tested for the control of an ochratoxigenic fungi (Penicillium nordicum) in a cheese matrix. The incorporation of L. buchneri cells resulted in thicker films with less luminosity than control films and colour alteration. Nevertheless, cells inclusion did not alter moisture content, water vapour permeability, mechanical properties, hydrophobicity and chemical structure of the films. Whey protein films were able to maintain the viability of L. buchneri UTAD104 cells in 105 CFU/mL after 30 days of storage at 25 \textdegreeC. When applied in cheese, films and coatings containing L. buchneri cells prevented fungal contamination for at least 30 days, while control cheeses with films and coatings either without LAB or with Lactobacillus casei UM3 (a strain without antifungal ability) showed fungal contamination during that period. Ochratoxin A was not found in cheeses treated with films and coatings containing L. buchneri UTAD104. Results showed that the inclusion of a LAB with antifungal properties in edible films and coatings can help to reduce or eliminate P. nordicum contamination in cheeses.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Ana Guimarães received support through grant SFRH/BD/103245/2014 from the Portuguese FCT.info:eu-repo/semantics/publishedVersio

Mapping of the fibroblast growth factors in human white adipose tissue.

CONTEXT: Fibroblast growth factors (FGFs) regulate the development of white adipose tissue (WAT). However, the secretion and cellular origin of individual FGFs in WAT as well as the influence of obesity are unknown. OBJECTIVE: Our objective was to map FGFs in human sc WAT, the cellular source, and association with obesity. DESIGN: Secretion, mRNA, and circulatory levels of FGFs in human abdominal sc WAT from nonobese and obese donors were examined by microarray, real-time quantitative PCR, and ELISA. The activity of FGFs in cultured human adipocytes was determined by phosphorylation assays. RESULTS: Expression of five FGFs (FGF1, FGF2, FGF7, FGF9, and FGF18) and FGF homologous factor (FHF2) was identified in WAT. Only FGF1 was released in a time-dependent manner from sc WAT, and fat cells were the major source of FGF1 secretion. FGF1 expression increased and FGF2 decreased during adipocyte differentiation. Furthermore, FGF1 was not secreted into the circulation. Although FGF1 levels were 2-fold increased in obesity, they were unaltered by weight reduction. Only FGF1 and FGF2 induced a marked concentration-dependent phosphorylation of p44/42 in cultured human adipocytes. CONCLUSIONS: Of the investigated FGFs, only FGF1 is secreted from sc WAT and predominantly so from the adipocyte fraction. The activity in adipocyte cultures and lack of secretion into the circulation suggest that FGF1 acts as an auto- or paracrine factor. FGF1 levels are increased in obesity but unaffected by weight reduction, suggesting a primary defect in obese individuals. In conclusion, FGF1 may play a superior role among the FGFs in sc WAT and obesity development