The Flavonol Quercitrin Hinders GSK3 Activity and Potentiates the Wnt/β-Catenin Signaling Pathway

The Wnt/β-catenin signaling pathway dictates cell proliferation and differentiation during embryonic development and tissue homeostasis. Its deregulation is associated with many pathological conditions, including neurodegenerative disease, frequently downregulated. The lack of efficient treatment for these diseases, including Alzheimer’s disease (AD), makes Wnt signaling an attractive target for therapies. Interestingly, novel Wnt signaling activating compounds are less frequently described than inhibitors, turning the quest for novel positive modulators even more appealing. In that sense, natural compounds are an outstanding source of potential drug leads. Here, we combine different experimental models, cell-based approaches, neuronal culture assays, and rodent behavior tests with Xenopus laevis phenotypic analysis to characterize quercitrin, a natural compound, as a novel Wnt signaling potentiator. We find that quercitrin potentiates the signaling in a concentration-dependent manner and increases the occurrence of the Xenopus secondary axis phenotype mediated by Xwnt8 injection. Using a GSK3 biosensor, we describe that quercitrin impairs GSK3 activity and increases phosphorylated GSK3β S9 levels. Treatment with XAV939, an inhibitor downstream of GSK3, impairs the quercitrin-mediated effect. Next, we show that quercitrin potentiates the Wnt3a-synaptogenic effect in hippocampal neurons in culture, which is blocked by XAV939. Quercitrin treatment also rescues the hippocampal synapse loss induced by intracerebroventricular injection of amyloid-β oligomers (AβO) in mice. Finally, quercitrin rescues AβO-mediated memory impairment, which is prevented by XAV939. Thus, our study uncovers a novel function for quercitrin as a Wnt/β-catenin signaling potentiator, describes its mechanism of action, and opens new avenues for AD treatments.     

Assessing T-Cell Immunity in Kidney Transplant Recipients with Absent Antibody Production after a 3rd Dose of the mRNA-1273 Vaccine

The vulnerable population of kidney transplant recipients (KTRs) are low responders to COVID-19 vaccines, so specific immune surveillance is needed. The interferon-gamma (IFN-γ) release assay (IGRA) is effective in assessing T cell-mediated immunity. We assessed SARS-CoV-2-directed T cell responses in KTRs with absent antibody production after a third dose of the mRNA-1273 vaccine, using two different IGRAs. A cohort of 57 KTRs, who were actively followed up, received a third dose of the mRNA-1273 vaccine. After the evaluation of humoral immunity to SARS-CoV-2, 14 seronegative patients were tested with two commercial IGRAs (SD Biosensor and Euroimmun). Out of 14 patients, one and three samples were positive by IGRAs with Euroimmun and SD Biosensor, respectively. The overall agreement between the two assays was 85.7% (κ = 0.444). In addition, multivariate linear regression analysis showed no statistically significant association between the IFN-γ concentration, and the independent variables analyzed (age, gender, years since transplant, total lymphocytes cells/mcl, CD3+ cells/mcl, CD3+ CD4+ cells/mcl, CD3+ CD8+ cells/mcl, CD19+ cells/mcl, CD3-CD16+CD56+ cells/mcl) (p > 0.01). In a vulnerable setting, assessing cellular immune response to complement the humoral response may be advantageous. Since the two commercial IGRAs showed a good agreement on negative samples, the three discordant samples highlight the need for further investigations.     

Evolution and resolution of long-term cardiac memory

BACKGROUND: Cardiac memory (CM) refers to T-wave changes induced by ventricular pacing or arrhythmia that accumulate in magnitude and duration with repeated episodes of abnormal activation. We report herein the kinetics of long-term CM and its association with the ventricular action potential. METHODS AND RESULTS: Dogs were paced from the ventricles at rates of 110 to 120 bpm for approximately 3 weeks. CM characterized by gradual sinus rhythm T vector rotation toward the paced QRS vector evolved in all dogs regardless of pacing site (left ventricular [LV] anterior apex or base, posterior LV, or right ventricular free wall). Cardiac hemodynamics and myocardial flow (microsphere studies) were unaltered by the pacing. Recovery time for the memory T wave to return to control increased with duration of the previous pacing. The protein synthesis inhibitor cycloheximide markedly (P<.05) and reproducibly attenuated evolution of CM. When pacing was performed from the atrium, CM did not occur. Standard microelectrode techniques were used to study action potential from the LV free wall of control and CM dogs. CM was associated with increased action potential duration in epicardial and endocardial but not midmyocardial cells, significantly altering the transmyocardial gradient for repolarization. CONCLUSIONS: CM is a dynamic process for which the final T vector is predicted by the paced QRS vector and which is associated with significant changes in epicardial and endocardial but not midmyocardial cell action potential duration, such that the transmural gradient of repolarization is altered. It is unaccompanied by evidence of altered hemodynamics or flow, requires a change in pathway of activation, and appears to require new protein synthesis.     

A double flow cytometric tag allows tracking of the dynamics of cell cycle progression of newborn Saccharomyces cerevisiae cells during balanced exponential growth

Studies on the dynamics of growth of single eukaryotic cells and their relationships with cell cycle regulations are generally carried out following cell synchronization procedures or, on a relatively low number of cells, by time-lapse studies. Establishment of both time-lapse studies and synchronous cell populations usually requires elaborate experimental efforts and is prone to perturb the physiological state of the cell. In this paper we use a new flow cytometric approach which allows, in asynchronous growing Saccharomyces cerevisiae populations, tagging of both the cell age and the cell protein content of a cohort of daughter cells at the different cell cycle set points. Since the cell protein content is a good estimation of the cell size, it is possible to follow the kinetics of the cell size increase during cell cycle progression. The experimental findings obtained indicate an exponential increase of the cell size during growth, that the daughter and the parent subpopulations grow with the same specific growth rate, that the average cell size increase rate of each individual cell is almost identical to the specific growth rate of the overall population and provide the opportunity to estimate the cell cycle length for the daughter cell population as well as the identification of the complex structure of asynchronously growing yeast populations.     

Effect of polythene film activated with enterocin EJ97 in combination with EDTA against Bacillus coagulans

Polythene films coated with the enterococcal bacteriocin enterocin EJ97 alone or in combination with EDTA were tested against Bacillus coagulans CECT 12. Bacteriocin activity was clearly enhanced by EDTA, as shown by viable staining and epifluorescence microscopy observation of treated cells. Activated films were tested in liquids from canned corn and canned peas inoculated with B. coagulans cell suspensions and stored at 4 °C and 20 °C for 24 h. The bacteriocin alone showed highest activity in samples stored at 4 °C, while the maximum performance of EDTA was observed at 20 °C. Films activated with a combination of both antimicrobials showed highest bactericidal activity at 4 °C. In liquid from canned corn and peas stored at 4 °C, the combined treatment reduced the concentrations of viable cells progressively over incubation time. Viable staining revealed an increase in the percentage of dead cells at 20 °C, avoiding proliferation of the bacilli. The bactericidal effect of the combined antimicrobial agents was higher in the liquid of canned peas than that of canned corn. The combined use of viable staining and classical viable cell counts allowed a better estimation of cell damage caused by the antimicrobial treatments.     

Coating-Activation and Antimicrobial Efficacy of Different Polyethylene Films with a Nisin-Based Solution

Antimicrobial packaging can be considered an extremely challenging technology that could have a significant impact on shelf-life extension and food safety of fresh meat and meat products. In this study, different commercial polyethylene films differing in vinyl acetate ethylene, erucamide contents, and oxygen permeability were used for the coating treatment with a nisin-based antimicrobial solution (NS). Detection and measurement of the activity of the NS was determined against different food spoilage bacteria. NS was then spread manually on food contact layer of different plastic films using coating rods providing thickness of 6, 40, 60, and 100 μm. The polyethylene films before and after treatment were analysed by atomic force microscopy (AFM). NS was active against Gram-positive bacteria and the best activity was obtained against Brochothrix thermosphacta. Viable staining and epifluorescence microscopy analysis of indicator strains in contact with activated plastic films showed that the effect of the film on the various indicator strains changed very much on the basis of both type of film and indicator strain. The highest numbers of lysed cells were shown by two polyethylene films that, according to the AFM and roughness parameters analyses, were characterized by significant increase or decrease of roughness after the coating treatment. AFM analysis showed that the homogeneity of the coating was much influenced by the type of plastic films used. In order to test the efficacy in food, portions of beef chuck tender slices were prepared and covered with the antimicrobial plastic films on both sides. After 1 h and 1, 7, and 12 days of storage at 4 °C the meat samples were analyzed by standard plate counting targeting spoilage associated microbial populations. The antimicrobial plastic films after 1 h of contact with the meat caused a significant reduction of lactic acid bacteria and B. thermosphacta. The most effective antimicrobial activity of films was shown against the same populations after 24 h of storage.     

Foam‐Mat Freeze‐Drying of Bifidobacterium longum RO175: Viability and Refrigerated Storage Stability

Foaming as a pretreatment was used prior to freeze‐drying of Bifidobacterium longum RO175 to investigate the potential acceleration of the drying rate and increase in microorganism viability after the process. A study on storage of foamed and nonfoamed freeze‐dried products at 4 °C completed this study. B. longum RO175 in foamed medium could be freeze‐dried in 1/7 to 1/4 of the time required for nonfoamed suspensions. In addition, foamed suspensions presented higher viability immediately after freeze‐drying (13.6% compared to 12.81 % or 11.46%, depending on the cryoprotective media). Refrigerated storage led to a reduction in B. longum RO175 viability for all tested protective agents (foamed and nonfoamed). No correlation between glass transition temperature and stability of probiotic powders was observed during storage. In addition, lower viability after 56 d of storage was observed for foamed materials, probably due to foam porous structure and higher hygroscopicity, and oxygen presence and moisture pickup during storage.