Real-time PCR analysis is a sensitive DNA quantification technique that has recently gained considerable attention in biotechnology, microbiology and molecular diagnostics. Although, the cycle-threshold (Ct) method is the present "gold standard", it is far from being a standard assay. Uniform reaction efficiency among samples is the most important assumption of this method. Nevertheless, some authors have reported that it may not be correct and a slight PCR efficiency decrease of about 4% could result in an error of up to 400% using the Ct method. This reaction efficiency decrease may be caused by inhibiting agents used during nucleic acid extraction or copurified from the biological sample. We propose a new method (Cy 0 ) that does not require the assumption of equal reaction efficiency between unknowns and standard curve. The Cy 0 method is based on the fit of Richards' equation to real-time PCR data by nonlinear regression in order to obtain the best fit estimators of reaction parameters. Subsequently, these parameters were used to calculate the Cy 0 value that minimizes the dependence of its value on PCR kinetic. The Ct, second derivative (Cp), sigmoidal curve fitting method (SCF) and Cy 0 methods were compared using two criteria: precision and accuracy. Our results demonstrated that, in optimal amplification conditions, these four methods are equally precise and accurate. However, when PCR efficiency was slightly decreased, diluting amplification mix quantity or adding a biological inhibitor such as IgG, the SCF, Ct and Cp methods were markedly impaired while the Cy 0 method gave significantly more accurate and precise results. Our results demonstrate that Cy 0 represents a significant improvement over the standard methods for obtaining a reliable and precise nucleic acid quantification even in sub-optimal amplification conditions overcoming the underestimation caused by the presence of some PCR inhibitors.

Social isolation has adverse effects on mental health, physical exercise, and dietary habits. This longitudinal observational study aimed to investigate the effects of mood states and exercise on nutritional choices, on 176 college students (92 males, 84 females; 23 ± 4 years old) during the COVID-19 lockdown. During 21 days, nutrition and exercise were daily monitored, and the mood states assessed. A factor analysis was used to reduce the number of nutritional variables collected. The relationships between exercise, mood and nutrition were investigated using a multivariate general linear model and a mediation model. Seven factors were found, reflecting different nutritional choices. Exercise was positively associated with fruit, vegetables and fish consumption (p = 0.004). Depression and quality of life were, directly and inversely, associated with cereals, legumes (p = 0.005; p = 0.004) and low-fat meat intake (p = 0.040; p = 0.004). Exercise mediated the effect of mood states on fruit, vegetables and fish consumption, respectively, accounting for 4.2% and 1.8% of the total variance. Poorer mood states possibly led to unhealthy dietary habits, which can themselves be linked to negative mood levels. Exercise led to healthier nutritional choices, and mediating the effects of mood states, it might represent a key measure in uncommon situations, such as home-confinement.

The adult gut microbiota contains trillions of microorganisms of thousands of different species. Only one third of gut microbiota are common to most people; the rest are specific and contribute to enhancing genetic variation. Gut microorganisms significantly affect host nutrition, metabolic function, immune system, and redox levels, and may be modulated by several environmental conditions, including physical activity and exercise. Microbiota also act like an endocrine organ and is sensitive to the homeostatic and physiological changes associated with training; in turn, exercise has been demonstrated to increase microbiota diversity, consequently improving the metabolic profile and immunological responses. On the other side, adaptation to exercise might be influenced by the individual gut microbiota that regulates the energetic balance and participates to the control of inflammatory, redox, and hydration status. Intense endurance exercise causes physiological and biochemical demands, and requires adequate measures to counteract oxidative stress, intestinal permeability, electrolyte imbalance, glycogen depletion, frequent upper respiratory tract infections, systemic inflammation and immune responses. Microbiota could be an important tool to improve overall general health, performance, and energy availability while controlling inflammation and redox levels in endurance athletes. The relationship among gut microbiota, general health, training adaptation and performance, along with a focus on sport supplements which are known to exert some influence on the microbiota, will be discussed.