Identifying the underlying child-eating behaviours that contribute to weight differences across growth has been a constant challenge. This report reviews the various literature approaches for assessing appetite regulation. In doing so, it attempts to understand how appetite control develops and determines the eating habits in early childhood, and its effects on children’s weight status. The interaction between homeostatic and hedonic mechanisms largely explains the appetite regulation process. Homeostatic mechanisms are mediated by the biological need to maintain the body’s energy reserves, increasing the motivation to eat. On the contrary, the hedonic mechanisms are mediated by food reward, increasing the craving for high-palatable foods and triggering the release of dopamine and serotonin. There are many biological methods (plasma measurements of hormones, like leptin, ghrelin and insulin) and behavioural evaluation methods of appetite. The Children’s Eating Behaviour Questionnaire is most commonly used, due to its adequate psychometric properties tested in several population settings. The development of eating behaviours begins in utero, and several determinants may contribute to a decrease in the ability to self-regulate dietary intake. Examples include genetic predisposition, the first taste experiences and the family environment, a key determinant in this process. Several eating behaviours contribute most to childhood obesity. Among them, are the external eating (eating by external stimuli, such as the mere presence of the food or its smell), food restriction (which may potentiate the uninhibited increased intake of the restricted foods) and emotional eating (intake due to emotional variations, especially negative feelings). These eating behaviours have been linked to childhood obesity.

Biofilm formation is recognized as the main virulence factor in a variety of chronic infections. In vitro evaluation of biofilm formation is often achieved by quantification of viable or total cells. However, these methods depend on biofilm disruption, which is often achieved by vortexing or sonication. In this study, we investigated the effects of sonication on the elimination of Staphylococcus epidermidis cell clusters from biofilms grown over time, and quantification was performed by three distinct analytical techniques. Even when a higher number of sonication cycles was used, some stable cell clusters remained in the samples obtained from 48- and 72-h-old biofilms, interfering with the quantification of sessile bacteria by plate counting. On the other hand, the fluorescence microscopy automatic counting system allowed proper quantification of biofilm samples that had undergone any of the described sonication cycles, suggesting that this is a more accurate method for assessing the cell concentration in S. epidermidis biofilms, especially in mature biofilms.

Staphylococcus epidermidis biofilm-related infections are a current concern within the medical community due to their high incidence and prevalence, particularly in patients with indwelling medical devices. Biofilm gene expression analysis by quantitative real-time PCR (qPCR) has been increasingly used to understand the role of biofilm formation in the pathogenesis of S. epidermidis infections. However, depending on the RNA extraction procedure, and cDNA synthesis and qPCR master mixes used, gene expression quantification can be suboptimal. We recently showed that some RNA extraction kits are not suitable for S. epidermidis biofilms, due to sample composition, in particular the presence of the extracellular matrix. In this work, we describe a custom RNA extraction assay followed by the evaluation of gene expression using different commercial reverse transcriptase kits and qPCR master mixes. Our custom RNA extraction assay was able to produce good quality RNA with reproducible gene expression quantification, reducing the time and the costs associated. We also tested the effect of reducing cDNA and qPCR reaction volumes and, in most of the cases tested, no significant differences were found. Finally, we titered the SYBR Green I concentrations in standard PCR master mixes and compared the normalized expression of the genes icaA, bhp, aap, psmβ1 and agrB using 4 distinct biofilm forming S. epidermidis strains to the results obtained with commercially available kits. The overall results demonstrated that despite some statistically, but not biologically significant differences observed, the customized qPCR protocol resulted in the same gene expression trend presented by the commercially available kits used.