c.376A>G, (p.Ser126Gly) Alpha-Galactosidase A mutation induces ER stress, unfolded protein response and reduced enzyme trafficking to lysosome: Possible relevance in the pathogenesis of late-onset forms of Fabry Disease.

Fabry Disease (FD) (OMIM 301500) is a metabolic X-linked inherited lysosomal storage disorder that results from the deficient activity of Alpha-Galactosidase A (Alpha-Gal), a lysosomal hydrolase that cleaves neutral glycosphingolipids with terminal N-linked galactosyl moieties, mainly globotriaosylceramides (Gb3). The enzyme, encoded by a 12-kb gene mapping on the long arm (Xq22.1 region) of the X chromosome, is constituted by a glycosylated subunit of approximately 55 kD, synthesized as an inactive precursor that undergoes maturation in endoplasmic reticulum (ER) and Golgi apparatus before being delivered to the lysosome to form a functional dimer. The gene is comprised of seven exons and, so far, >1000 different mutations have been described as associated to FD (www.dbfgp.org/dbFgp/fabry/FabryGP.htm). Clinical phenotypes are divided in two main classes, classic or non-classic, based on clinical and biochemical findings. Non-classic FD, usually recognized as late-onset forms with oligosymptomatic phenotype, presents with symptoms restricted solely to cardiocytes, kidneys or brain associated to missense misfolding mutations. In the group of the non-classic FD, special attention should be given to patients carrying the c.376A > G (p.Ser126Gly) mutation. The lack of clear experimental evidences on its pathogenetic role, despite the clinical pictures of the patients with severe ischaemic lesions, renal involvement and acroparesthesias, led many authors to classify this mutation as inconsistent, non-pathogenetic, and consequently not eligible to the current pharmacological treatments for FD. To shed light on the cellular processes affected by this mutation and to assess if the biochemical pathways involved with, could really have a significant pathogenetic impact, we studied the mutation in silico and in COS-7 and HEK 293 cell models. We found p.Ser126Gly, even retaining both high degree of synthesis and residual activity, is mostly stacked into the ER inducing unfolded protein response (UPR) with reduced trafficking to the lysosome. These data strongly suggest that p.Ser126Gly could trigger a pathogenetic mechanism different from the classic and well assessed increased turnover with loss of biological activity described for other missense mutations. This mechanism seems mainly related to a negative gain of function, with ER retention and UPR activation and could lead, via inflammation and/or apoptosis, to irreversible cell damage.

Acceptance of recommended vaccinations during pregnancy: a cross-sectional study in Southern Italy.

Background: Vaccine administration is a recommended, safe, and effective measure to protect pregnant women against vaccine-preventable diseases (VPDs). Despite available guidance, maternal immunization rates for vaccination against influenza and with the reduced antigen content tetanus-diphtheria-acellular pertussis vaccine (Tdap) in Italy remain incredibly low. The primary goal of the study was to explore what Italian pregnant women knew about VPDs and immunization during pregnancy and what factors affected their decision to be vaccinated. Methods: This cross-sectional study took place between October 2021 and April 2022 in the Southern part of Italy. All consecutive pregnant women, from those attending the selected facilities on randomly selected days, were approached to request participation. The inclusion criteria for participation were age ≥18 years, the ability to understand, speak, and read Italian, and being pregnant at any gestational age. The questionnaire, using a combination of checkboxes and free text answers, consisted of 32 items divided into five parts and lasted ~10 min. Results: The results showed that 61% knew that the influenza vaccine is recommended and 48.7% knew that influenza could be risky during pregnancy; 74.1% wrongly reported that the Measles-Mumps-Rubella (MMR) vaccine is recommended during pregnancy. Seven out of 10 pregnant women believed that strong evidence supported the safety of vaccinations during pregnancy, and more than half (55.6%) thought they were at increased risk of severe illness with COVID-19. Women in the sample believed that vaccines received during pregnancy pose a risk of adverse events to the unborn child with a median value of 6 (IQR 3-9), on a scale ranging from 1 to 10. Similarly, the fear of contracting pertussis and influenza during pregnancy showed a median value of 6 (IQR 3-9) and 5 (IQR 3-8), respectively. Only 21.1% and 36.5% of women received influenza and Tdap vaccines during pregnancy. Conclusion: Unrealistic risk perception with a negative attitude toward vaccines in pregnancy and a low percentage of vaccinated pregnant women confirm the urgency of training women to make informed choices to increase overall vaccine uptake.

Apigenin and Luteolin Regulate Autophagy by Targeting NRH-Quinone Oxidoreductase 2 in Liver Cells.

Dietary flavonoids stimulate autophagy and prevent liver dysfunction, but the upstream signaling pathways triggered by these compounds are not well understood. Certain polyphenols bind directly to NRH-quinone oxidoreductase 2 (NQO2) and inhibit its activity. NQO2 is highly expressed in the liver, where it participates in quinone metabolism, but recent evidence indicates that it may also play a role in the regulation of oxidative stress and autophagy. Here, we addressed a potential role of NQO2 in autophagy induction by flavonoids. The pro-autophagic activity of seven flavonoid aglycons correlated perfectly with their ability to inhibit NQO2 activity, and flavones such as apigenin and luteolin showed the strongest activity in all assays. The silencing of NQO2 strongly reduced flavone-induced autophagic flux, although it increased basal LC3-II levels in HepG2 cells. Both flavones induced AMP kinase (AMPK) activation, while its reduction by AMPK beta (PRKAB1) silencing inhibited flavone-induced autophagy. Interestingly, the depletion of NQO2 levels by siRNA increased the basal AMPK phosphorylation but abrogated its further increase by apigenin. Thus, NQO2 contributes to the negative regulation of AMPK activity and autophagy, while its targeting by flavones releases pro-autophagic signals. These findings imply that NQO2 works as a flavone receptor mediating autophagy and may contribute to other hepatic effects of flavonoids.

Bergamot Polyphenols Boost Therapeutic Effects of the Diet on Non-Alcoholic Steatohepatitis (NASH) Induced by "Junk Food": Evidence for Anti-Inflammatory Activity.

Wrong alimentary behaviors and so-called "junk food" are a driving force for the rising incidence of non-alcoholic fatty liver disease (NAFLD) among children and adults. The "junk food" toxicity can be studied in "cafeteria" (CAF) diet animal model. Young rats exposed to CAF diet become obese and rapidly develop NAFLD. We have previously showed that bergamot (Citrus bergamia Risso et Poiteau) flavonoids, in the form of bergamot polyphenol fraction (BPF), effectively prevent CAF diet-induced NAFLD in rats. Here, we addressed if BPF can accelerate therapeutic effects of weight loss induced by a normocaloric standard chow (SC) diet. 21 rats fed with CAF diet for 16 weeks to induce NAFLD with inflammatory features (NASH) were divided into three groups. Two groups were switched to SC diet supplemented or not with BPF (CAF/SC±BPF), while one group continued with CAF diet (CAF/CAF) for 10 weeks. BPF had no effect on SC diet-induced weight loss, but it accelerated hepatic lipid droplets clearance and reduced blood triglycerides. Accordingly, BPF improved insulin sensitivity, but had little effect on leptin levels. Interestingly, the inflammatory parameters were still elevated in CAF/SC livers compared to CAF/CAF group after 10 weeks of dietary intervention, despite over 90% hepatic fat reduction. In contrast, BPF supplementation decreased hepatic inflammation by reducing interleukin 6 (Il6) mRNA expression and increasing anti-inflammatory Il10, which correlated with fewer Kupffer cells and lower inflammatory foci score in CAF/SC+BPF livers compared to CAF/SC group. These data indicate that BPF mediates a specific anti-inflammatory activity in livers recovering from NASH, while it boosts lipid-lowering and anti-diabetic effects of the dietary intervention.