The Landscape of microRNAs in βCell: Between Phenotype Maintenance and Protection

Diabetes mellitus is a group of heterogeneous metabolic disorders characterized by chronic hyperglycaemia mainly due to pancreatic β cell death and/or dysfunction, caused by several types of stress such as glucotoxicity, lipotoxicity and inflammation. Different patho-physiological mechanisms driving β cell response to these stresses are tightly regulated by microRNAs (miRNAs), a class of negative regulators of gene expression, involved in pathogenic mechanisms occurring in diabetes and in its complications. In this review, we aim to shed light on the most important miRNAs regulating the maintenance and the robustness of β cell identity, as well as on those miRNAs involved in the pathogenesis of the two main forms of diabetes mellitus, i.e., type 1 and type 2 diabetes. Additionally, we acknowledge that the understanding of miRNAs-regulated molecular mechanisms is fundamental in order to develop specific and effective strategies based on miRNAs as therapeutic targets, employing innovative molecules.     

The GAUGAA Motif Is Responsible for the Binding between circSMARCA5 and SRSF1 and Related Downstream Effects on Glioblastoma Multiforme Cell Migration and Angiogenic Potential

Circular RNAs (circRNAs) are a large class of RNAs with regulatory functions within cells. We recently showed that circSMARCA5 is a tumor suppressor in glioblastoma multiforme (GBM) and acts as a decoy for Serine and Arginine Rich Splicing Factor 1 (SRSF1) through six predicted binding sites (BSs). Here we characterized RNA motifs functionally involved in the interaction between circSMARCA5 and SRSF1. Three different circSMARCA5 molecules (Mut1, Mut2, Mut3), each mutated in two predicted SRSF1 BSs at once, were obtained through PCR-based replacement of wild-type (WT) BS sequences and cloned in three independent pcDNA3 vectors. Mut1 significantly decreased its capability to interact with SRSF1 as compared to WT, based on the RNA immunoprecipitation assay. In silico analysis through the “Find Individual Motif Occurrences” (FIMO) algorithm showed GAUGAA as an experimentally validated SRSF1 binding motif significantly overrepresented within both predicted SRSF1 BSs mutated in Mut1 (q-value = 0.0011). U87MG and CAS-1, transfected with Mut1, significantly increased their migration with respect to controls transfected with WT, as revealed by the cell exclusion zone assay. Immortalized human brain microvascular endothelial cells (IM-HBMEC) exposed to conditioned medium (CM) harvested from U87MG and CAS-1 transfected with Mut1 significantly sprouted more than those treated with CM harvested from U87MG and CAS-1 transfected with WT, as shown by the tube formation assay. qRT-PCR showed that the intracellular pro- to anti-angiogenic Vascular Endothelial Growth Factor A (VEGFA) mRNA isoform ratio and the amount of total VEGFA mRNA secreted in CM significantly increased in Mut1-transfected CAS-1 as compared to controls transfected with WT. Our data suggest that GAUGAA is the RNA motif responsible for the interaction between circSMARCA5 and SRSF1 as well as for the circSMARCA5-mediated control of GBM cell migration and angiogenic potential.     

Interplay between Humoral and CLA+ T Cell Response against Candida albicans in Psoriasis

Candida albicans (CA) infections have been associated with psoriasis onset or disease flares. However, the integrated immune response against this fungus is still poorly characterized in psoriasis. We studied specific immunoglobulins in plasma and the CA response in cocultures of circulating memory CD45RA⁻ cutaneous lymphocyte antigen (CLA)^+/− T cell with autologous epidermal cells from plaque and guttate psoriasis patients (cohort 1, n = 52), and also healthy individuals (n = 17). A complete proteomic profile was also evaluated in plaque psoriasis patients (cohort 2, n = 114) regarding their anti-CA IgA levels. Increased anti-CA IgA and IgG levels are present in the plasma from plaque but not guttate psoriasis compared to healthy controls. CA cellular response is confined to CLA⁺ T cells and is primarily Th17. The levels of anti-CA IgA are directly associated with CLA⁺ Th17 response in plaque psoriasis. Proteomic analysis revealed distinct profiles in psoriasis patients with high anti-CA IgA. C-C motif chemokine ligand 18, chitinase-3-like protein 1 and azurocidin were significantly elevated in the plasma from plaque psoriasis patients with high anti-CA levels and severe disease. Our results indicate a mechanism by which Candida albicans exposure can trigger a clinically relevant IL-17 response in psoriasis. Assessing anti-CA IgA levels may be useful in order to evaluate chronic psoriasis patients.     

BRAF Gene and Melanoma: Back to the Future

As widely acknowledged, 40–50% of all melanoma patients harbour an activating BRAF mutation (mostly BRAF V600E). The identification of the RAS–RAF–MEK–ERK (MAP kinase) signalling pathway and its targeting has represented a valuable milestone for the advanced and, more recently, for the completely resected stage III and IV melanoma therapy management. However, despite progress in BRAF-mutant melanoma treatment, the two different approaches approved so far for metastatic disease, immunotherapy and BRAF+MEK inhibitors, allow a 5-year survival of no more than 60%, and most patients relapse during treatment due to acquired mechanisms of resistance. Deep insight into BRAF gene biology is fundamental to describe the acquired resistance mechanisms (primary and secondary) and to understand the molecular pathways that are now being investigated in preclinical and clinical studies with the aim of improving outcomes in BRAF-mutant patients.     

Prophylaxis and Treatment against Klebsiella pneumoniae: Current Insights on This Emerging Anti-Microbial Resistant Global Threat

Klebsiella pneumoniae (Kp) is an opportunistic pathogen and the leading cause of healthcare-associated infections, mostly affecting subjects with compromised immune systems or suffering from concurrent bacterial infections. However, the dramatic increase in hypervirulent strains and the emergence of new multidrug-resistant clones resulted in Kp occurrence among previously healthy people and in increased morbidity and mortality, including neonatal sepsis and death across low- and middle-income countries. As a consequence, carbapenem-resistant and extended spectrum β-lactamase-producing Kp have been prioritized as a critical anti-microbial resistance threat by the World Health Organization and this has renewed the interest of the scientific community in developing a vaccine as well as treatments alternative to the now ineffective antibiotics. Capsule polysaccharide is the most important virulence factor of Kp and plays major roles in the pathogenesis but its high variability (more than 100 different types have been reported) makes the identification of a universal treatment or prevention strategy very challenging. However, less variable virulence factors such as the O-Antigen, outer membrane proteins as fimbriae and siderophores might also be key players in the fight against Kp infections. Here, we review elements of the current status of the epidemiology and the molecular pathogenesis of Kp and explore specific bacterial antigens as potential targets for both prophylactic and therapeutic solutions.     

High-Fat Diet Leads to Reduced Protein O-GlcNAcylation and Mitochondrial Defects Promoting the Development of Alzheimer’s Disease Signatures

The disturbance of protein O-GlcNAcylation is emerging as a possible link between altered brain metabolism and the progression of neurodegeneration. As observed in brains with Alzheimer’s disease (AD), flaws of the cerebral glucose uptake translate into reduced protein O-GlcNAcylation, which promote the formation of pathological hallmarks. A high-fat diet (HFD) is known to foster metabolic dysregulation and insulin resistance in the brain and such effects have been associated with the reduction of cognitive performances. Remarkably, a significant role in HFD-related cognitive decline might be played by aberrant protein O-GlcNAcylation by triggering the development of AD signature and mitochondrial impairment. Our data support the impairment of total protein O-GlcNAcylation profile both in the brain of mice subjected to a 6-week high-fat-diet (HFD) and in our in vitro transposition on SH-SY5Y cells. The reduction of protein O-GlcNAcylation was associated with the development of insulin resistance, induced by overfeeding (i.e., defective insulin signaling and reduced mitochondrial activity), which promoted the dysregulation of the hexosamine biosynthetic pathway (HBP) flux, through the AMPK-driven reduction of GFAT1 activation. Further, we observed that a HFD induced the selective impairment of O-GlcNAcylated-tau and of O-GlcNAcylated-Complex I subunit NDUFB8, thus resulting in tau toxicity and reduced respiratory chain functionality respectively, highlighting the involvement of this posttranslational modification in the neurodegenerative process.     

The Role of Circulating Biomarkers in Peripheral Arterial Disease

Peripheral arterial disease (PAD) of the lower extremities is a chronic illness predominantly of atherosclerotic aetiology, associated to traditional cardiovascular (CV) risk factors. It is one of the most prevalent CV conditions worldwide in subjects >65 years, estimated to increase greatly with the aging of the population, becoming a severe socioeconomic problem in the future. The narrowing and thrombotic occlusion of the lower limb arteries impairs the walking function as the disease progresses, increasing the risk of CV events (myocardial infarction and stroke), amputation and death. Despite its poor prognosis, PAD patients are scarcely identified until the disease is advanced, highlighting the need for reliable biomarkers for PAD patient stratification, that might also contribute to define more personalized medical treatments. In this review, we will discuss the usefulness of inflammatory molecules, matrix metalloproteinases (MMPs), and cardiac damage markers, as well as novel components of the liquid biopsy, extracellular vesicles (EVs), and non-coding RNAs for lower limb PAD identification, stratification, and outcome assessment. We will also explore the potential of machine learning methods to build prediction models to refine PAD assessment. In this line, the usefulness of multimarker approaches to evaluate this complex multifactorial disease will be also discussed.     

Cyclic Nigerosyl-Nigerose as Oxygen Nanocarrier to Protect Cellular Models from Hypoxia/Reoxygenation Injury: Implications from an In Vitro Model

Heart failure (HF) prevalence is increasing among the aging population, and the mortality rate remains unacceptably high despite improvements in therapy. Myocardial ischemia (MI) and, consequently, ischemia/reperfusion injury (IRI), are frequently the basis of HF development. Therefore, cardioprotective strategies to limit IRI are mandatory. Nanocarriers have been proposed as alternative therapy for cardiovascular disease. Controlled reoxygenation may be a promising strategy. Novel nanocarriers, such as cyclic nigerosyl-nigerose (CNN), can be innovative tools for oxygen delivery in a controlled manner. In this study we analyzed new CNN-based formulations as oxygen nanocarriers (O₂-CNN), and compared them with nitrogen CNN (N₂-CNN). These different CNN-based formulations were tested using two cellular models, namely, cardiomyoblasts (H9c2), and endothelial (HMEC) cell lines, at different concentrations. The effects on the growth curve during normoxia (21% O₂, 5% CO₂ and 74% N₂) and their protective effects during hypoxia (1% O₂, 5% CO₂ and 94% N₂) and reoxygenation (21% O₂, 5% CO₂ and 74% N₂) were studied. Neither O₂-CNN nor N₂-CNN has any effect on the growth curve during normoxia. However, O₂-CNN applied before hypoxia induces a 15–30% reduction in cell mortality after hypoxia/re-oxygenation when compared to N₂-CNN. O₂-CNN showed a marked efficacy in controlled oxygenation, which suggests an interesting potential for the future medical application of soluble nanocarrier systems for MI treatment.     

Tau Exon 10 Inclusion by PrPC through Downregulating GSK3β Activity

Tau protein is largely responsible for tauopathies, including Alzheimer’s disease (AD), where it accumulates in the brain as insoluble aggregates. Tau mRNA is regulated by alternative splicing, and inclusion or exclusion of exon 10 gives rise to the 3R and 4R isoforms respectively, whose balance is physiologically regulated. In this sense, one of the several factors that regulate alternative splicing of tau is GSK3β, whose activity is inhibited by the cellular prion protein (PrP^C), which has different physiological functions in neuroprotection and neuronal differentiation. Moreover, a relationship between PrP^C and tau expression levels has been reported during AD evolution. For this reason, in this study we aimed to analyze the role of PrP^C and the implication of GSK3β in the regulation of tau exon 10 alternative splicing. We used AD human samples and mouse models of PrP^C ablation and tau overexpression. In addition, we used primary neuronal cultures to develop functional studies. Our results revealed a paralleled association between PrP^C expression and tau 4R isoforms in all models analyzed. In this sense, reduction or ablation of PrP^C levels induces an increase in tau 3R/4R balance. More relevantly, our data points to GSK3β activity downstream from PrP^C in this phenomenon. Our results indicate that PrP^C plays a role in tau exon 10 inclusion through the inhibitory capacity of GSK3β.