Stability study of Prulifloxacin and Ulifloxacin in human plasma by HPLC–DAD

A new and specific HPLC–DAD method for the direct determination of Prulifloxacin and its active metabolite, Ulifloxacin, in human plasma has been developed. Plasma samples were analysed after a simple solid phase extraction (SPE) clean-up using a new HILIC stationary phase based high-performance liquid chromatography (HPLC) column and an ammonium acetate buffer (5?mM, pH 5.8)/acetonitrile (both with 1% Et₃N, v/v) mobile phase in isocratic elution mode, with Danofloxacin as the internal standard. Detection was performed using DAD from 200 to 500?nm and quantitative analyses were carried out at 278?nm. The LOQ of the method was 1?μg/mL of the cited analytes and the calibration curve showed a good linearity up to 25?μg/mL. For both analytes the precision (RSD%) and the trueness (bias%) of the method fulfil with International Guidelines. The method was applied for stability studies, at three QC concentration levels, in human plasma samples stored at different temperature of?+?25,?+?4 and ?20?°C in order to evaluate plasma stability profiles.     

β-glucans: ex vivo inflammatory and oxidative stress results after pasta intake

Background

It is well known that Mediterranean Diet can positively influence the health of each individual, in particular it is know that fibers have an important role. However, in Mediterranean cities most people do not have a close adherence to Mediterranean diet. Thus, in our study, we considered fibers like β-glucans that have been added to pasta with a percentage of 6 %. Our study aimed to evaluate the capacity of β-glucans intake on oxidative stress and inflammation in a cohort of middle aged slightly overweight subjects.

Methods

We used a longitudinal study design. The study lasted 30 days during which time, each participant acted with no food restriction. Participants underwent morning fasting blood venous sample for blood chemistry and other biological parameters at the beginning of the study and after 30 days of pasta supplemented with 6 % of β-glucan intake 4 times a week. We performed anthropometric, biochemical, oxidative stress and cytokine analysis at the beginning and the end of study.

Results

After the 30 days of pasta intake we obtained a significant decrease of LDL-cholesterol, IL-6 and AGEs levels.

Conclusion

The results confirmed a capacity of β-glucans intake to lower oxidative stress. Additional longitudinal observation on community-based cohorts are needed to confirm these data and investigate the biological mechanisms through which effects are induced, and to fully explore the therapeutic potential of β-glucans.

     

A Structural Overview of the Vertebrate Prion Proteins

Among the diseases caused by protein misfolding is the family associated with the prion protein (PrP). This is a small extracellular membrane-anchored molecule of yet unknown function. Understanding how PrP folds both into its cellular and pathological forms is thought to be crucial for explaining protein misfolding in general and the specific role of PrP in disease. Since the first structure determination, an increasing number of structural studies of PrP have become available, showing that the protein is formed by a flexible N-terminal region and a highly conserved globular C-terminal domain. We review here the current knowledge on PrP structure. We focus on vertebrate PrPs and analyse in detail the similarities and the differences among the coordinates of the C-terminal domain of PrP from different species, in search for understanding the mechanism of disease-causing mutations and the molecular bases of species barrier.Key Words: amyloid, NMR, prion, scrapie, structure, X-ray     

Haemagglutinin substitutions N125D,D127E,D222G and R223Q improve replicative fitness and vaccine effectiveness of an A/H1N1pdm09 live attenuated influenza vaccine virus by enhancing α-2,6 receptor binding

During 2013–14 and 2015–16, A/H1N1pdm09 live attenuated influenza vaccine (LAIV) viruses replicated inefficiently in primary human nasal epithelial cells (hNEC). This led to reduced vaccine effectiveness (VE) in quadrivalent formulations, mediated by inter-strain competition. By mutating the haemagglutinin (HA) protein, we aimed to enhance hNEC replication of a novel A/H1N1pdm09 vaccine strain to overcome competition and improve VE. Combinations of N125D, D127E, D222G and R223Q substitutions were introduced to the HA protein of A/Slovenia/2903/2015 (A/SLOV15). A/SLOV15 S13, containing all four HA substitutions, produced approximately 1000-fold more virus than parental V1 during hNEC infection. Immunogenicity in ferrets was increased by approximately 10-fold, without compromising yield in eggs or antigenic match to wild-type (wt) reference strains. Despite S13 and V1 being antigenically similar, only S13 protected ferrets from wt virus shedding and fever post-challenge. Crucially, these data suggested that enhanced fitness allowed S13 to overcome inter-strain competition in quadrivalent LAIV (QLAIV). This improved efficacy was later validated by real-world VE data. S13 displayed increased binding avidity to a mammalian-like α-2,6 receptor analogue (6-SLN), relative to V1, while maintaining avian-like 3-SLN avidity. In silico modelling of the HA receptor binding site revealed additional interactions in the S13:6-SLN binding network and a mild increase in 6-SLN binding energy, indicating a possible mechanism for increased α-2,6 receptor-binding avidity. These data confirm that rational HA mutagenesis can be used to optimise hNEC replication and VE for A/H1N1pdm09 LAIV viruses.     

Autocrine abscisic acid mediates the UV-B-induced inflammatory response in human granulocytes and keratinocytes

UV-B is an abiotic environmental stress in both plants and animals. Abscisic acid (ABA) is a phytohormone regulating fundamental physiological functions in plants, including response to abiotic stress. We previously demonstrated that ABA is an endogenous stress hormone also in animal cells. Here, we investigated whether autocrine ABA regulates the response to UV-B of human granulocytes and keratinocytes, the cells involved in UV-triggered skin inflammation. The intracellular ABA concentration increased in UV-B-exposed granulocytes and keratinocytes and ABA was released into the supernatant. The UV-B-induced production of NO and of reactive oxygen species (ROS), phagocytosis, and cell migration were strongly inhibited in granulocytes irradiated in the presence of a monoclonal antibody against ABA. Moreover, presence of the same antibody strongly inhibited release of NO, prostaglandin E2 (PGE(2)), and tumor necrosis factor-α (TNF-α) by UV-B irradiated keratinocytes. Lanthionine synthetase C-like protein 2 (LANCL2) is required for the activation of the ABA signaling pathway in human granulocytes. Silencing of LANCL2 in human keratinocytes by siRNA was accompanied by abrogation of the UV-B-triggered release of PGE(2), TNF-α, and NO and ROS production. These results indicate that UV-B irradiation induces ABA release from human granulocytes and keratinocytes and that autocrine ABA stimulates cell functions involved in skin inflammation.