Circulating Small EVs miRNAs as Predictors of Pathological Response to Neo-Adjuvant Therapy in Breast Cancer Patients

Neo-adjuvant therapy (NAT) is increasingly used in the clinic for the treatment of breast cancer (BC). Pathological response to NAT has been associated with improved patients’ survival; however, the current techniques employed for assessing the tumor response have significant limitations. Small EVs (sEVs)-encapsulated miRNAs have emerged as promising new biomarkers for diagnosis and prediction. Therefore, our study aims to explore the predictive value of these miRNAs for the pathological response to NAT in BC. By employing bioinformatic tools, we selected a set of miRNAs and evaluated their expression in plasma sEVs and BC biopsies. Twelve miRNAs were identified in sEVs, of which, miR-21-5p, 221-3p, 146a-5p and 26a-5p were significantly associated with the Miller–Payne (MP) pathological response to NAT. Moreover, miR-21-5p, 146a-5p, 26a-5p and miR-24-3p were independent as predictors of MP response to NAT. However, the expression of these miRNAs showed no correlation between sEVs and tissue samples, indicating that the mechanisms of miRNA sorting into sEVs still needs to be elucidated. Functional analysis of miRNA target genes and drug interactions revealed that candidate miRNAs and their targets, can be regulated by different NAT regimens. This evidence supports their role in governing the patients’ therapy response and highlights their potential use as prediction biomarkers.     

Interaction of miR-155 with Human Serum Albumin: An Atomic Force Spectroscopy,Fluorescence, FRET,and Computational Modelling Evidence

This study investigated the interaction between Human Serum Albumin (HSA) and microRNA 155 (miR-155) through spectroscopic, nanoscopic and computational methods. Atomic force spectroscopy together with static and time-resolved fluorescence demonstrated the formation of an HSA/miR-155 complex characterized by a moderate affinity constant (K_A in the order of 10⁴ M⁻¹). Förster Resonance Energy Transfer (FRET) experiments allowed us to measure a distance of (3.9 ± 0.2) nm between the lone HSA Trp214 and an acceptor dye bound to miR-155 within such a complex. This structural parameter, combined with computational docking and binding free energy calculations, led us to identify two possible models for the structure of the complex, both characterized by a topography in which miR-155 is located within two positively charged pockets of HSA. These results align with the interaction found for HSA and miR-4749, reinforcing the thesis that native HSA is a suitable miRNA carrier under physiological conditions for delivering to appropriate targets.     

Binding of Gamma-Glutamyl Transferase to TLR4 Signalling Allows Tissue Factor Activation in Monocytes

Gamma-glutamyl transferase (GGT) is involved in the progression of atherosclerosis, since its enzymatic activity promotes the generation of reactive oxygen species (ROS). Besides, GGT may act as a prothrombotic factor by inducing tissue factor (TF) expression, independently of its enzymatic activity. The aim of this study was to assess whether GGT-induced TF stimulation was a consequence of binding to toll-like receptor 4 (TLR4) expressed on monocytes, the precursors of macrophages and foam cells which colocalize with GGT activity within atherosclerotic plaques. Experiments were performed in human peripheral blood mononuclear cells (PBMCs), THP-1 cells (a monocytic cellular model), and HEK293 cells, which were genetically modified to study the activation of TLR4. TF procoagulant activity was assessed by a one-stage clotting time test, and TF protein expression was estimated by western blot. Human recombinant (hr) GGT protein increased TF procoagulant activity and protein expression in both PBMCs and THP-1 cells. The GGT-induced TF stimulation was prevented by cellular pretreatment with TLR4/NF-κB inhibitors (LPS-Rs, CLI-095, and BAY-11-7082), and HEK293 cells lacking TLR4 confirmed that TLR4 is essential for GGT-induced activation of NF-κB. In conclusion, hrGGT induced TF expression in monocytes through a cytokine-like mechanism that involved the activation of TLR4/NF-κB signaling.     

Biochemical Characterization of Pyranose Oxidase from Streptomyces canus—Towards a Better Understanding of Pyranose Oxidase Homologues in Bacteria

Pyranose oxidase (POx, glucose 2-oxidase; EC 1.1.3.10, pyranose:oxygen 2-oxidoreductase) is an FAD-dependent oxidoreductase and a member of the auxiliary activity (AA) enzymes (subfamily AA3_4) in the CAZy database. Despite the general interest in fungal POxs, only a few bacterial POxs have been studied so far. Here, we report the biochemical characterization of a POx from Streptomyces canus (ScPOx), the sequence of which is positioned in a separate, hitherto unexplored clade of the POx phylogenetic tree. Kinetic analyses revealed that ScPOx uses monosaccharide sugars (such as d-glucose, d-xylose, d-galactose) as its electron-donor substrates, albeit with low catalytic efficiencies. Interestingly, various C- and O-glycosides (such as puerarin) were oxidized by ScPOx as well. Some of these glycosides are characteristic substrates for the recently described FAD-dependent C-glycoside 3-oxidase from Microbacterium trichothecenolyticum. Here, we show that FAD-dependent C-glycoside 3-oxidases and pyranose oxidases are enzymes belonging to the same sequence space.     

Modeling RTT Syndrome by iPSC-Derived Neurons from Male and Female Patients with Heterogeneously Severe Hot-Spot MECP2 Variants

Rett syndrome caused by MECP2 variants is characterized by a heterogenous clinical spectrum accounted for in 60% of cases by hot-spot variants. Focusing on the most frequent variants, we generated in vitro iPSC-neurons from the blood of RTT girls with p.Arg133Cys and p.Arg255*, associated to mild and severe phenotype, respectively, and of an RTT male harboring the close to p.Arg255*, p.Gly252Argfs*7 variant. Truncated MeCP2 proteins were revealed by Western blot and immunofluorescence analysis. We compared the mutant versus control neurons at 42 days for morphological parameters and at 120 days for electrophysiology recordings, including girls’ isogenic clones. A precocious reduced morphological complexity was evident in neurons with truncating variants, while in p.Arg133Cys neurons any significant differences were observed in comparison with the isogenic wild-type clones. Reduced nuclear size and branch number show up as the most robust biomarkers. Patch clamp recordings on mature neurons allowed the assessment of cell biophysical properties, V-gated currents, and spiking pattern in the mutant and control cells. Immature spiking, altered cell capacitance, and membrane resistance of RTT neurons, were particularly pronounced in the Arg255* and Gly252Argfs*7 mutants. The overall results indicate that the specific markers of in vitro cellular phenotype mirror the clinical severity and may be amenable to drug testing for translational purposes.     

KDM2A and KDM3B as Potential Targets for the Rescue of F508del-CFTR

Cystic fibrosis (CF) is caused by mutations in the gene encoding of the cystic fibrosis transmembrane conductance regulator (CFTR), an anion-selective plasma membrane channel that mainly regulates chloride transport in a variety of epithelia. More than 2000 mutations, most of which presumed to be disease-relevant, have been identified in the CFTR gene. The single CFTR mutation F508del (deletion of phenylalanine in position 508) is present in about 90% of global CF patients in at least one allele. F508del is responsible for the defective folding and processing of CFTR, failing to traffic to the plasma membrane and undergoing premature degradation via the ubiquitin–proteasome system. CFTR is subjected to different post-translational modifications (PTMs), and the possibility to modulate these PTMs has been suggested as a potential therapeutic strategy for the functional recovery of the disease-associated mutants. Recently, the PTM mapping of CFTR has identified some lysine residues that may undergo methylation or ubiquitination, suggesting a competition between these two PTMs. Our work hypothesis moves from the idea that favors methylation over ubiquitination, e.g., inhibiting demethylation could be a successful strategy for preventing the premature degradation of unstable CFTR mutants. Here, by using a siRNA library against all the human demethylases, we identified the enzymes whose downregulation increases F508del-CFTR stability and channel function. Our results show that KDM2A and KDM3B downregulation increases the stability of F508del-CFTR and boosts the functional rescue of the channel induced by CFTR correctors.     

Potential of the Stromal Matricellular Protein Periostin as a Biomarker to Improve Risk Assessment in Prostate Cancer

Prostate cancer (PCa) ranges from indolent to aggressive tumors that may rapidly progress and metastasize. The switch to aggressive PCa is fostered by reactive stroma infiltrating tumor foci. Therefore, reactive stroma-based biomarkers may potentially improve the early detection of aggressive PCa, ameliorating disease classification. Gene expression profiles of PCa reactive fibroblasts highlighted the up-regulation of genes related to stroma deposition, including periostin and sparc. Here, the potential of periostin as a stromal biomarker has been investigated on PCa prostatectomies by immunohistochemistry. Moreover, circulating levels of periostin and sparc have been assessed in a low-risk PCa patient cohort enrolled in active surveillance (AS) by ELISA. We found that periostin is mainly expressed in the peritumoral stroma of prostatectomies, and its stromal expression correlates with PCa grade and aggressive disease features, such as the cribriform growth. Moreover, stromal periostin staining is associated with a shorter biochemical recurrence-free survival of PCa patients. Interestingly, the integration of periostin and sparc circulating levels into a model based on standard clinico-pathological variables improves its performance in predicting disease reclassification of AS patients. In this study, we provide the first evidence that circulating molecular biomarkers of PCa stroma may refine risk assessment and predict the reclassification of AS patients.     

Adipose Tissue Dysfunction and Obesity-Related Male Hypogonadism

Obesity is a chronic illness associated with several metabolic derangements and comorbidities (i.e., insulin resistance, leptin resistance, diabetes, etc.) and often leads to impaired testicular function and male subfertility. Several mechanisms may indeed negatively affect the hypothalamic–pituitary–gonadal health, such as higher testosterone conversion to estradiol by aromatase activity in the adipose tissue, increased ROS production, and the release of several endocrine molecules affecting the hypothalamus–pituitary–testis axis by both direct and indirect mechanisms. In addition, androgen deficiency could further accelerate adipose tissue expansion and therefore exacerbate obesity, which in turn enhances hypogonadism, thus inducing a vicious cycle. Based on these considerations, we propose an overview on the relationship of adipose tissue dysfunction and male hypogonadism, highlighting the main biological pathways involved and the current therapeutic options to counteract this condition.     

ILB®, a Low Molecular Weight Dextran Sulphate,Restores Glutamate Homeostasis,Amino Acid Metabolism and Neurocognitive Functions in a Rat Model of Severe Traumatic Brain Injury

In a previous study, we found that administration of ILB^®, a new low molecular weight dextran sulphate, significantly improved mitochondrial functions and energy metabolism, as well as decreased oxidative/nitrosative stress, of brain tissue of rats exposed to severe traumatic brain injury (sTBI), induced by the closed-head weight-drop model of diffused TBI. Using aliquots of deproteinized brain tissue of the same animals of this former study, we here determined the concentrations of 24 amino acids of control rats, untreated sTBI rats (sacrificed at 2 and 7 days post-injury) and sTBI rats receiving a subcutaneous ILB^® administration (at the dose levels of 1, 5 and 15 mg/kg b.w.) 30 min post-impact (sacrificed at 2 and 7 days post-injury). Additionally, in a different set of experiments, new groups of control rats, untreated sTBI rats and ILB^®-treated rats (administered 30 min after sTBI at the dose levels of 1 or 5 mg/kg b.w.) were studied for their neurocognitive functions (anxiety, locomotor capacities, short- and long-term memory) at 7 days after the induction of sTBI. Compared to untreated sTBI animals, ILB^® significantly decreased whole brain glutamate (normalizing the glutamate/glutamine ratio), glycine, serine and γ-aminobutyric acid. Furthermore, ILB^® administration restored arginine metabolism (preventing nitrosative stress), levels of amino acids involved in methylation reactions (methionine, L-cystathionine, S-adenosylhomocysteine), and N-acetylaspartate homeostasis. The macroscopic evidences of the beneficial effects on brain metabolism induced by ILB^® were the relevant improvement in neurocognitive functions of the group of animals treated with ILB^® 5 mg/kg b.w., compared to the marked cognitive decline measured in untreated sTBI animals. These results demonstrate that ILB^® administration 30 min after sTBI prevents glutamate excitotoxicity and normalizes levels of amino acids involved in crucial brain metabolic functions. The ameliorations of amino acid metabolism, mitochondrial functions and energy metabolism in ILB^®-treated rats exposed to sTBI produced significant improvement in neurocognitive functions, reinforcing the concept that ILB^® is a new effective therapeutic tool for the treatment of sTBI, worth being tested in the clinical setting.     

Platelet Derivatives and the Immunomodulation of Wound Healing

Besides their primary role in hemostasis, platelets contain a plethora of immunomodulatory molecules that profoundly affect the entire process of wound repair. Therefore, platelet derivatives, such as platelet-rich plasma or platelet lysate, have been widely employed with promising results in the treatment of chronic wounds. Platelet derivatives provide growth factors, cytokines, and chemokines targeting resident and immigrated cells belonging to the innate and adaptive immune system. The recruitment and activation of neutrophils and macrophages is critical for pathogen clearance in the early phase of wound repair. The inflammatory response begins with the release of cytokines, such as TGF-β, aimed at damping excessive inflammation and promoting the regenerative phase of wound healing. Dysregulation of the immune system during the wound healing process leads to persistent inflammation and delayed healing, which ultimately result in chronic wound. In this review, we summarize the role of the different immune cells involved in wound healing, particularly emphasizing the function of platelet and platelet derivatives in orchestrating the immunological response.