Polypyrimidine Tract Binding Protein (PTB) is an intensely studied RNA binding protein involved in several post-transcriptional regulatory events of gene expression. Initially described as a pre-mRNA splicing regulator, PTB is now widely accepted as a multifunctional protein shuttling between nucleus and cytoplasm. Accordingly, PTB can interact with selected RNA targets, structural elements and proteins. There is increasing evidence that PTB and its paralog PTBP2 play a major role as repressors of alternatively spliced exons, whose transcription is tissue-regulated. In addition to alternative splicing, PTB is involved in almost all steps of mRNA metabolism, including polyadenylation, mRNA stability and initiation of protein translation. Furthermore, it is well established that PTB recruitment in internal ribosome entry site (IRES) activates the translation of picornaviral and cellular proteins. Detailed studies of the structural properties of PTB have contributed to our understanding of the mechanism of RNA binding by RNA Recognition Motif (RRM) domains. In the present review, we will describe the structural properties of PTB, its paralogs and co-factors, the role in post-transcriptional regulation and actions in cell differentiation and pathogenesis. Defining the multifunctional roles of PTB will contribute to the understanding of key regulatory events in gene expression.

Bile acids (BA) play a pivotal role in cholesterol metabolism. Their blood concentration has also been proposed as new prognostic and diagnostic indicator of hepatobiliary, intestinal, and cardiovascular disease. Liquid chromatography tandem mass spectrometry (LC–MS/MS) currently represents the gold standard for analysis of BA profile in biological samples. We report here development and validation of a LC–MS/MS technique for simultaneously quantifying 15 BA species in serum samples. We also established a reference range for adult healthy subjects (n = 130) and performed a preliminary evaluation of in vitro and in vivo interference. The method displayed good linearity, with high regression coefficients (>0.99) over a range of 5 ng/mL (lower limit of quantification, LLOQ) and 5000 ng/mL for all analytes tested. The accuracies were between 85–115%. Both intra- and inter-assay imprecision was <10%. The recoveries ranged between 92–110%. Each of the tested BA species (assessed on three concentrations) were stable for 15 days at room temperature, 4 °C, and −20 °C. The in vitro study did not reveal any interference from triglycerides, bilirubin, or cell-free hemoglobin. The in vivo interference study showed that pools obtained from hyper-cholesterolemic patients and hyper-bilirubinemic patients due to post-hepatic jaundice for benign cholestasis, cholangiocarcinoma and pancreatic head tumors had clearly distinct patterns of BA concentrations compared with a pool obtained from samples of healthy subjects. In conclusion, this study proposes a new suitable candidate method for identification and quantitation of BA in biological samples and provides new insight into a number of variables that should be taken into account when investigating pathophysiological changes of BA in human diseases.

Aromatic amino acid decarboxylase (AADC) deficiency is a severe inherited recessive neurotransmitter disorder caused by an impairment in dopamine synthesis due to the lack/modification of AADC, the enzyme converting l ‐dopa to dopamine. Patients exhibit severe movement disorders and neurodevelopmental delay, with a high risk of premature mortality. Given the lack of a reliable model for the disease, we developed a dopa decarboxylase knockout model using CRISPR/Cas9 technology in the SH‐SY5Y neuroblastoma cell line. This model showed a deficiency in AADC protein and activity, with an altered dopamine metabolites profile (low homovanillic acid and high 3‐O‐methyldopa) and a modified expression of key enzymes, such as dopamine beta‐hydroxylase and monoamine oxidases, which are involved in the catecholamine pathway. We then transfected the DDC ‐KO cells with two AADC catalytic variants, R347Q and L353P, which resulted in loss‐of‐function and an altered profile of dopamine metabolites. By combining several structural approaches (X‐ray crystallography, molecular dynamics, small angle X‐ray scattering, dynamic light scattering, and spectroscopy), we determined that both variants alter the flexibility of the structural element to which they belong, whose integrity is essential for catalysis. This change causes a mispositioning of essential residues at the active site, leading, in turn, to an unproductive external aldimine, identifying the molecular basis for the loss‐of‐function. Overall, the DDC ‐KO model recapitulates some key features of AADC deficiency, is useful to study the molecular basis of the disease, and represents an ideal system for small molecule screening regarding specific enzyme defects, paving the way for a precision therapeutic approach.

Studies investigating the potential role of circulating bile acids (BAs) as diagnostic biomarkers for cholangiocarcinoma (CCA) are sparse and existing data do not adjust for confounding variables. Furthermore, the mechanism by which BAs affect the expression of the oncogenic mucin 5AC (MUC5AC) has never been investigated. We performed a case–control study to characterise the profile of circulating BAs in patients with CCA (n = 68) and benign biliary disease (BBD, n = 48) with a validated liquid chromatography–tandem mass spectrometry technique. Odd ratios (OR) for CCA associations were calculated with multivariable logistic regression models based on a directed acyclic graph structure learning algorithm. The most promising BAs were then tested in an in vitro study to investigate their interplay in modulating MUC5AC expression. The total concentration of BAs was markedly higher in patients with CCA compared with BBD controls and accompanied by a shift in BAs profile toward a higher proportion of primary conjugated BAs (OR = 1.50, CI: 1.14 to 1.96, p = 0.003), especially taurochenodeoxycholic acid (TCDCA, OR = 42.29, CI: 3.54 to 504.63, p = 0.003) after multiple adjustments. Western blot analysis of secreted MUC5AC in human primary cholangiocytes treated with primary conjugated BAs or with TCDCA alone allowed us to identify a novel 230 kDa isoform, possibly representing a post-translationally modified MUC5AC specie.

Post-translational modifications (PTMs) are crucial chemical alterations occurring on proteins post-synthesis, impacting various cellular processes. During viral infections, PTMs are shown to play a multitude of roles in viral replication, host interaction, and immune evasion. Thus, these modifications can influence infectivity, with direct impact on the anti-viral host immune responses and potentially viral adaptation across species. This field is still scarcely explored, whilst understanding PTMs is not only important to advance the knowledge of virus pathology but also potentially to provide insights for vaccine development. In this review, we attempt to summarize the latest findings mainly published over the last 10 years, focusing on the roles of PTMs involved in virus infection and anti-viral immune responses, in the context of relevant human respiratory infections: influenza A virus (IAV), respiratory syncytial virus (RSV), and SARS-CoV-2. We decided to concentrate on these three viruses because they currently represent a global health problem due to recurrent outbreaks and pandemic potential. A deeper characterization of the PTMs may help in understanding virus–host interaction with possible implications on curative strategies. Further, we will report on cutting-edge technologies to study in vitro virus infection in different cellular-based systems. In particular, we describe and discuss the application of 2D and 3D lung organoid cell-culture systems as in vitro models to mimic respiratory environments and to study the PTMs in a controlled setting. Finally, we will discuss the importance of PTMs in the context of next-generation vaccine design, especially for their potential role to offer effective protection against respiratory viruses.