Contribution of vitamin D3 and thiols status to the outcome of COVID-19 disease in Italian pediatric and adult patients.

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARSCoV-2), was declared a global pandemic by the World Health Organization (WHO) on March 2020, causing unprecedented disease with million deaths across the globe, mostly adults. Indeed, children accounted for only a few percent of cases. Italy was the first Western country struck by the COVID-19 epidemic. Increasing age, which is one of the principal risk factors for COVID-19 mortality, is associated with declined glutathione (GSH) levels. Over the last decade, several studies demonstrated that both vitamin D (VD) and GSH have immunomodulatory properties. To verify the association between VD, GSH and the outcome of COVID-19 disease, we conducted a multicenter retrospective study in 35 children and 128 adult patients with COVID-19. Our study demonstrated a hypovitaminosis D in COVID-19 patients, suggesting a possible role of low VD status in increasing the risk of COVID-19 infection and subsequent hospitalization. In addition, we find a thiol disturbance with a GSH depletion associated to the disease severity. In children, who fortunately survived, both VD and GSH levels at admission were higher than in adults, suggesting that lower VD and thiols levels upon admission may be a modifiable risk factor for adverse outcomes and mortality in hospitalized patients with COVID-19.

Study of the Association between Thiols and Oxidative Stress Markers in Children with Obesity.

Obesity has reached epidemic proportions, and the World Health Organization defined childhood overweight and obesity as a noncommunicable disease that represents the most serious public health challenges of the twenty-first century. Oxidative stress, defined as an imbalance between oxidants and antioxidants causing an impairment of the redox signals, is linked to the development of metabolic diseases. In addition, reactive oxygen species generated during metabolic disorder could increase inflammation, causing the development of insulin resistance, diabetes, and cardiovascular disease. We analyze serum levels of cysteine (Cys), cysteinyl-glycine (Cys-Gly), homocysteine (Hcy), and glutathione (GSH), and other markers of oxidative stress, such as thiobarbituric acid reactive substances (T-BARS), 8-isoprostane, and protein carbonyl in our children with obesity. Total antioxidant status was also determined. We found lower GSH and Cys-Gly levels, and higher Hcy and oxidative stress markers levels. We also found a positive correlation between Body Mass Index (BMI), Cys, GSH, and Hcy levels, between insulin and Cys levels, and between BMI and the homeostasis model assessment-estimated insulin resistance (HOMA-IR) with 8-isoprostane levels. Finally, we found a correlation between age and GSH and Cys levels. The deficiency of GSH could be restored by dietary supplementation with GSH precursors, supplying an inexpensive approach to oppose oxidative stress, thus avoiding obesity complications.

Soluble Isoform of Suppression of Tumorigenicity 2 (ST2) Biomarker in a Large Cohort of Healthy Pediatric Population: Determination of Reference Intervals.

Introduction: Only little data exists on ST2 reference intervals in healthy pediatric populations despite the high importance of this biomarker in adults with heart failure. The aim of the study was to assess the reference intervals of ST2 in a wide healthy pediatric cohort. Methods: We evaluated the serum concentrations of ST2 biomarker in 415 healthy pediatric subjects referred to our analysis laboratory. Subjects were categorized according to age (i.e., 0−6 (n = 79), 7−11 (n = 142) and 12−18 years (n = 191)) and sex. They were not suffering from any cardiac disorders, metabolic disorders, lung diseases, autoimmune disorders or malignancies. A written consent was obtained for each individual. No duplicate patients were included in the analysis and the presence of outliers was investigated. Reference intervals (Mean and central 95% confidence intervals) were determined. Results: Three outliers have been identified and removed from the analysis (60.0, 64.0 and 150.2 ng/mL). A total of 412 subjects were therefore included. The mean value for the whole population was 15.8 ng/mL (2.4−36.4 ng/mL). Males present a significantly higher mean concentration compared to females (17.2 versus 14.4 ng/mL, p = 0.001). A significant trend toward higher ST2 values with age was also observed, but for males only (r = 0.43, p < 0.0001). If considering age partitions, only males of 12−18 years (mean = 21.7 ng/mL) had significantly higher ST2 values compared to the other groups (ranging from 11.9 for males 0−6 years to 15.2 for females 12−18 years; p < 0.0001). Conclusions: We described age and sex-specific reference intervals for ST2 in a large healthy pediatric population. We found that ST2 values differ between sexes if considering all participants. A significant increase in ST2 with age was also observed, but only for males of 12−18 years.

Cerebrospinal Fluid Levels of AFP and hCG: Validation of the Analytical Method and Application in the Diagnosis of Central Nervous System Germ Cell Tumors.

The determination of Human Chorionic Gonadotropin (hCG) and Alpha Fetoprotein (AFP) levels on serum and amniotic fluid plays a fundamental role in the diagnosis and follow-up of specific physiological or pathological conditions (e.g., pregnancy, threat of abortion or germ cell tumors). Recently, the quantification of hCG and AFP in other biological fluids has gained great attention to support the diagnosis, prognosis and follow-up of neoplastic diseases deriving from trophoblastic cells, such as germinomas. Most of the commercial kits for hCG and AFP assays are developed to be used on biological fluids such as serum/plasma and/or urine by manufacturing companies. The aim of this work was to evaluate the suitability of the analytical method certified for the use on serum, and/or amniotic fluid for the quantification of hCG and AFP in cerebrospinal fluid, carrying out an internal validation protocol. The data reported here show that the automated immunochemical method is fit for quantification of hCG and AFP in cerebrospinal fluid (CSF), allowing selective and specific diagnosis of secreting germ cell tumors. This is confirmed by the positive correlation between elevated levels of hCG or AFP and the diagnosis of brain tumors.

Proteomic profiling of ATM kinase proficient and deficient cell lines upon blockage of proteasome activity.

Ataxia Telangiectasia Mutated (ATM) protein kinase is a key effector in the modulation of the functionality of some important stress responses, including DNA damage and oxidative stress response, and its deficiency is the hallmark of Ataxia Telangiectasia (A-T), a rare genetic disorder. ATM modulates the activity of hundreds of target proteins, essential for the correct balance between proliferation and cell death. The aim of this study is to evaluate the phenotypic adaptation at the protein level both in basal condition and in presence of proteasome blockage in order to identify the molecules whose level and stability are modulated through ATM expression. We pursued a comparative analysis of ATM deficient and proficient lymphoblastoid cells by label-free shotgun proteomic experiments comparing the panel of proteins differentially expressed. Through a non-supervised comparative bioinformatic analysis these data provided an insight on the functional role of ATM deficiency in cellular carbohydrate metabolism's regulation. This hypothesis has been demonstrated by targeted metabolic fingerprint analysis SRM (Selected Reaction Monitoring) on specific thermodynamic checkpoints of glycolysis. This article is part of a Special Issue entitled: Translational Proteomics.

Protein repertoire impact of Ubiquitin-Proteasome System impairment: insight into the protective role of beta-estradiol.

The Ubiquitin-Proteasome System (UPS) and the Autophagy-Lysosome Pathways (ALP) are key mechanisms for cellular homeostasis sustenance and protein clearance. A wide number of Neurodegenerative Diseases (NDs) are tied with UPS impairment and have been also described as proteinopathies caused by aggregate-prone proteins, not efficiently removed by proteasome. Despite the large knowledge on proteasome biological role, molecular mechanisms associated with its impairment are still blur. We have pursued a comprehensive proteomic investigation to evaluate the phenotypic rearrangements in protein repertoires associated with a UPS blockage. Different functional proteomic approaches have been employed to tackle UPS impairment impact on human NeuroBlastoma (NB) cell lines responsive to proteasome inhibition by Epoxomicin. 2-Dimensional Electrophoresis (2-DE) separation combined with Mass Spectrometry and Shotgun Proteomics experiments have been employed to design a thorough picture of protein profile. Unsupervised meta-analysis of the collected proteomic data revealed that all the identified proteins relate each other in a functional network centered on beta-estradiol. Moreover we showed that treatment of cells with beta-estradiol resulted in aggregate removal and increased cell survival due to activation of the autophagic pathway. Our data may provide the molecular basis for the use of beta-estradiol in neurodegenerative disorders by induction of protein aggregate removal.

New insights into neuroblastoma cisplatin resistance: a comparative proteomic and meta-mining investigation.

Neuroblastoma is one of the most aggressive solid tumors in the childhood. Therapy resistance to anticancer drugs represents the major limitation to the effectiveness of clinical treatment. To better understand the mechanisms underlying cisplatin resistance, we performed a comparative proteomic study of the human neuroblastoma cell line SH-SY5Y and its cisplatin resistant counterpart by both the classical 2-DE electrophoresis coupled to mass spectrometry and the more innovative label-free nLC-MS(E). The differentially expressed proteins were classified by bioinformatic tools according to their biological functions and their involvement in several cellular processes. Moreover, a meta-mining investigation of protein ontologies was also performed on available data from previously published proteomics studies to highlight the modulation of significant cellular pathways, which may regulate the sensitivity of neuroblastoma to cisplatin. In particular, we hypothesized a major role of the transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway. Confocal microscopy experiments, enzyme assay, and Western blotting of proteins regulated by Nrf2 provided evidences that this pathway, playing a protective role in normal cells, may represent a potential novel target to control cisplatin resistance in neuroblastoma.

W7FW14F apomyoglobin amyloid aggregates-mediated apoptosis is due to oxidative stress and AKT inactivation caused by Ras and Rac.

We have previously reported that addition of prefibrillar aggregates (PFAs) derived from W7FW14F apomyoglobin mutant to NIH-3T3 cells affects their viability. In this article, we have found that cytotoxicity induced by PFAs in NIH 3T3 and SH-SY5Y human neuroblastoma cells was due to early activation of apoptotic cell death dependent from a caspase-3- and -9-mediated mitochondrial pathway. A time-dependent increase of intracellular ROS and an about twofold decrease of mitochondrial localization of scavenger protein MnSOD was found. The use of the anti-oxidant agent N-acetyl-cysteine (NAC) antagonized both the increase of intracellular ROS and apoptosis induced by PFAs. PFAs caused an about 60% increase of the activity of both Ras and Erk-1/2 at 30 and 45 min while they were restored to basal levels at later time points. This effect was paralleled by a time-dependent decrease of the activity of the survival enzyme Akt. Effects similar to those on Ras activity were also recorded on the activity of the stress involved small GTP binding protein Rac that was about 75% increased after 30 min but resumed to basal levels at later time points. This effect was paralleled by a time-dependent activation of p38 kinase activity and HSP-70 expression. The use of both the ras farnesyltransferase inhibitor tipifarnib and the Rac geranyl-geranyltransferase GGTI-298, but not of the MEK-1 inhibitor U0126 partially antagonized the effects of PFAs on apoptosis occurrence. On the other hand, the PI3K/Akt inhibitor LY 294002 potentiated apoptosis induced by PFAs. Our results indicate a role for Ras and Rac in the induction of both intracellular ROS increased levels and apoptosis mediated by PFAs and disclose a new scenario of intervention in neurodegenerative diseases.

Proteasome inhibitors therapeutic strategies for cancer.

Aberrations in the Ubiquitin-Proteasome System (UPS) have been recently connected to the pathogenesis of several human protein degradation disorders (e.g., cancer and neurodegenerative diseases), so that proteasome is now considered an important target for drug discovery. Small molecules able to inhibit and modulate UPS have been, in fact, described as novel tools for a new approach in anti-cancer therapy. In particular Proteasome Inhibitors (PIs), blocking activation of nuclear factor-kappa B (NF-kB), trigger a decreased cellular proliferation and angiogenic cytokine production, induce cell death and inhibit tumor cell adhesion to stroma. Furthermore, several studies have demonstrated that PIs potentiate the activity of other anti-cancer treatment, in part by down-regulating chemoresistance pathways. Therefore pharmacologic, preclinical, and clinical data suggested the use of PIs in anticancer strategies, for their potential therapeutic relevance in the treatment of cancer and inflammatory-related diseases. This review focuses on recent advances in the development of PIs anticancer agents highlighting both novel patented compounds and novel therapeutic protocol of intervention.