Search

In spermatozoa, the nuclear F-actin supports the acroplaxome, a subacrosomal structure involved in the correct exposure of several acrosomal membrane proteins; among them, the glycoprotein IZUMO1 is the major protein involved in sperm-oocyte fusion. Nuclear F-actin is also involved in sperm head shaping and chromosome compartmentalization. To date, few notions regarding the bivalent role of F-actin on sperm chromatin organization and IZUMO1 positioning have been reported. In our work, we characterized subcellular organization of F-actin in human high- and low-quality spermatozoa (A- and B-SPZ), respectively, showing that F-actin over-expression in sperm head of B-SPZ affected IZUMO1 localization. A correct IZUMO1 repositioning following in vitro induction of F-actin depolymerization, by cytochalasin D treatment, occurred. Interestingly, F-actin depolymerization was also associated with a correct acrosome repositioning, thus to favor a proper acrosome reaction onset, with changes in sperm nuclear size parameters and histone acetylation rate reaching high-quality conditions. In conclusion, the current work shows a key role of F-actin in the control of IZUMO1 localization as well as chromatin remodeling and acetylation events.

There is a great debate regarding the usefulness and efficacy of this medication in anti-viral treatment: chloroquine is not included in the panel recommended for HIV treatment, while its modest effect in the therapy of human virus infection is reported for chronic hepatitis C These findings did not allow for its inclusion in the standardised therapeutic protocols for hepatitis C patients The benefits of chloroquine therapy strongly depend on: (i) the age of the patient;(ii) the clinical presentation and (iii) the stage of the COVID-19 disease Noteworthy, the use of this drug is contraindicated in some conditions, particularly the glucose-6-phosphate dehydrogenase (G6PD) deficiency The latter is a condition that has not been deeply taken into account particularly when, on the web, there has been a "viral" spread of news emphasizing the safe use and the free availability of chloroquine Therefore, before establishing that the use of chloroquine in the treatment of SARS-CoV-2 could represent a good option in light of such announcements, we should not miss the following important information that limit the friendly access to the drug: (a) G6PD is a housekeeping enzyme that, in the red blood cell (RBC), guarantees the production of NADPH, which is required to preserve glutathione in the reduced state (GSH), (b) G6PD deficiency shows marked genetic heterogeneity In this regard, we have reported more than 186 mutations, (c) G6PD deficiency is a necessary but not sufficient condition for the occurrence of clinical manifestations: the incidence of clinically evident favism in a group of randomly selected enzymopenic individuals resulted to be less than 30%, (d) the estimated number of G6PD-deficient individuals is close to 400 million people worldwide, while the number of pathogenic gene variants is generally concentrated in a small group of variations that can be easily detected in reference laboratories, and (e) as the rate of deficient RBCs in heterozygote carriers is not predictable a priori, potential individual clinical complications can strongly be proportional to the fraction of the number of G6PD-imbalanced RBCs Moreover, the majority of G6PD-deficient subjects do not show clinical manifestations in the steady state and the condition remains undetected until they are exposed to an exogenous haemolytic trigger such as bacterial or viral infections, ingestion of fava beans (favism) or drugs, mainly the hydroxychloroquine

The analysis of sterol profile consists in the qualitative and quantitative determination of cholesterol and non-cholesterol sterols. Non-cholesterol sterols include the cholesterol precursors in de novo biosynthesis pathway, cholesterol catabolites and plant sterols absorbed from diet. The sterol profile analysis can be performed on different biological matrices, i.e., plasma/serum, whole blood, erythrocyte membranes, tissues and cells, depending on the clinical and/or experimental purpose. The reference method for the sterol profile analysis is the gas-chromatography coupled with mass spectrometry, although new methods based on liquid-chromatography or direct mass spectrometry have been developed. The sterol profile analysis represents a fundamental tool for the biochemical diagnosis of the congenital defects of cholesterol metabolism. In addition, some non-cholesterol sterols have been validated as surrogate markers of de novo synthesis and intestinal absorption of cholesterol. Therefore, the sterol profile analysis has been used for the evaluation of cholesterol homeostasis in different diseases. This review focuses on these applications and our clinical and experimental findings. In the last nine years, we identified 18 new cases of the cholesterol metabolism defects and we found that cholesterol metabolism is impaired in patients with cystic fibrosis.

In the original article, there was a mistake in the order of Figures 1 and 2 as published. The figures are given in the correct order below. The authors apologize for the error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.

Background Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of autoantibodies to multiple self-antigens, including heat shock proteins (HSP). Because of the increased expression of HSP90 and abnormal immune responses to it in SLE, we investigated whether an HSP90 DNA vaccine could modulate the development and clinical manifestations of SLE in lupus-prone mice. Methods (NZB x NZW)F1 (NZB/W) mice were vaccinated with DNA constructs encoding HSP90 or control plasmids or vehicle. The mice were then monitored for survival, circulating anti-dsDNA autoantibodies, and immune phenotypes. Renal disease was evaluated by immunohistochemistry and by the measurement of proteinuria. Results Vaccination with HSP90 DNA reduced lupus disease manifestations and prolonged the survival of NZB/W mice. The protective effects of the HSP90 DNA vaccine associated with the induction of tolerogenic dendritic cells (DCs) and an expansion of T regulatory cells (Tregs). Conclusions The beneficial effects of DNA vaccination with HSP90 in murine SLE support the possibility of HSP90-based therapeutic modalities of intervention in SLE.

The causative gene of Cystic Fibrosis (CF) is the Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Although monogenic, CF has a complex genotype - phenotype relationship mainly originated by the high number of CFTR variants, the challenging mutational analysis, the incomplete functional knowledge and the variable clinical outcome of most variants, the action of modifier genes. This complexity affects diagnosis, prognosis and precision therapy. A correct and complete genetic analysis is crucial for all these aspects. The Consensus Document 2019 on the genetic analysis of CF (1S) provides a guidance to practitioners for the appropriate request and proper use of genetic testing in CF, as well as for the correct interpretation of the test result. It is to be hoped that the broad sharing achieved between the scientific societies involved can standardize the approach at national level and can improve the usefulness of the information provided to users, as part of a continuous process of improvement.

More than 2000 CFTR mutations have been identified so far, but only few of them are clearly defined as CF-causing based on functional studies. We present a case of a rare mutation, the G970D, that has been shown using transfected cDNA in HEK293 cells to be sensitive to Ivacaftor. However, a similar missense mutant, G970R in the same codon, was found to be sensitive to potentiators in vitro, but not in vivo due to splicing alteration. Thus, we used several basic research methodologies to evaluate if this patient was eligible for treatment with Ivacaftor. Materials and methods 1) nasal epithelial cells (HNEC) were collected from patients to evaluate the effect of mutations on splicing by RT-PCR assay, 2) HNEC were expanded and polarized for evaluation of CFTR function by Ussing chamber system 3) the use of a minigene system was used to confirm in vitro the splicing pattern. Results Firstly, we used in silico tool to predict the physio-pathological effect of mutations, confirming that the G970R completely abolishes the canonical 5’ splice donor site of exon 17 resulting in a likely retention of intron 17. On the contrary, the G970D predicted not to affect splicing. This prediction was confirmed by RT-PCR analysis of mRNA extracted from HNEC cells and from in vitro minigene assay. Finally, the functional behavior of CFTR, from HNEC bearing the G970D, was evaluated by short-circuit recordings. The cells responded to cAMP agonist with an increase in trans-epithelial current and this current was nearly doubled by stimulation with Ivacaftor. Moreover, in cells that were also incubated with VX-809 (1 μM) for 24 hours, CFTR function was significantly enhanced, with a proportional increase of both cAMP- and potentiator-dependent responses. Conclusions Our results show that the G970R actually disrupts the RNA splicing, thus leading to a severely altered CFTR protein. This event explains the lack of success of treatment with potentiator. In contrast, the G970D does not alter RNA splicing. The resulting G970D mutation affects the gating and trafficking of the CFTR channel that can be targeted with VX-770 and VX-809. These results represent the evidence needed to justify the treatment of the patient with these drugs. Finally, our study is an interesting example of the precision medicine approach by emphasizing the role of appropriate in vitro studies, in this case focused on RNA analysis, to fully characterize the effects of rare CFTR mutations. We thank Telethon Foundation (TMLGCBX16TT) and Ministero della Salute (Rome, Italy) L.548/93 for the regional research funding quote 2008-2015.