Retrospective analysis of seroprevalence in a cohort of university students of Rome (Italy) between September 2020 and July 2021.

BACKGROUND: the reopening of Universities in a COVID-19 pandemic context represented a potential source of virus transmission among students. OBJECTIVES: to measure the SARS-CoV-2 real circulation among university students attending the University of Rome 'Niccolò Cusano' by seroprevalence analysis. DESIGN: retrospective study based on a point-of-care (POC) SARS-CoV-2 rapid qualitative serological screening performed on asymptomatic students attending the University. SETTING AND PARTICIPANTS: between September 2020 to July 2021 at Niccolò Cusano University, 9,588 SARS-CoV-2 lateral-flow rapid qualitative antibody assays were performed on a total of 2,423 asymptomatic students. Among them, 389 individuals with compulsory attendance were tested every 10 days for a minimum observational period of 7 months. MAIN OUTCOME MEASURES: the prevalence of SARS-CoV-2 IgM/IgG antibodies was estimated at more levels. It was calculated: 1. the number of positive cases detected among the total number of students tested during the screening period; 2. the cumulative seroprevalence over the time, and the seroprevalence distribution over the months; 3. the duration of seropositivity after SARS-CoV-2 infection in the known previous infected students repeatedly tested. RESULTS: a total of 112 participants had a SARS-CoV-2 positive IgG and/or IgM antibodies test, 39 of them with a documented history of previous infection. In the remaining 73 cases, 24 were confirmed with an external quantitative serological analysis and identified as individuals with unknown previous SARS-CoV-2 exposure, 17 resulted false positive and 32 subjects were excluded. The total seroprevalence was 2.6% (95%CI 2.0%-3.3%) and among the 63 confirmed seropositive cases, 75% had detectable IgG antibodies, 3% had IgM antibodies, and 22% were positive for both IgM and IgG antibodies. In the 389 repeatedly-tested students, 36 students were positive to SARS-CoV-2 antibodies, 14 with unknown previous infection, and 22 with known previous infection. Among these, 50% retained immune memory up to 4 months post infection and 27% of cases retained seropositivity up to 7 months. CONCLUSIONS: the data collected has been useful to measure a real epidemiological rate of the virus spread in a cohort of students in Italy as well as to obtain information on the antibodies seropositivity duration in individuals with previous infection.

Nonthyroidal illness syndrome (NTIS) in severe COVID-19 patients: role of T3 on the Na/K pump gene expression and on hydroelectrolytic equilibrium.

BACKGROUND: Nonthyroidal Illness Syndrome (NTIS) can be detected in many critical illnesses. Recently, we demonstrated that this condition is frequently observed in COVID-19 patients too and it is correlated with the severity the disease. However, the exact mechanism through which thyroid hormones influence the course of COVID-19, as well as that of many other critical illnesses, is not clear yet and treatment with T4, T3 or a combination of both is still controversial. Aim of this study was to analyze body composition in COVID-19 patients in search of possible correlation with the thyroid function. METHODS AND FINDINGS: We report here our experience performed in 74 critically ill COVID-19 patients hospitalized in the intensive care unit (ICU) of our University Hospital in Rome. In these patients, we evaluated the thyroid hormone function and body composition by Bioelectrical Impedance Analysis (BIA) during the acute phase of the disease at admission in the ICU. To examine the effects of thyroid function on BIA parameters we analyzed also 96 outpatients, affected by thyroid diseases in different functional conditions. We demonstrated that COVID-19 patients with low FT3 serum values exhibited increased values of the Total Body Water/Free Fat Mass (TBW/FFM) ratio. Patients with the lowest FT3 serum values had also the highest level of TBW/FFM ratio. This ratio is an indicator of the fraction of FFM as water and represents one of the best-known body-composition constants in mammals. We found an inverse correlation between FT3 serum values and this constant. Reduced FT3 serum values in COVID-19 patients were correlated with the increase in the total body water (TBW), the extracellular water (ECW) and the sodium/potassium exchangeable ratio (Nae:Ke), and with the reduction of the intracellular water (ICW). No specific correlation was observed in thyroid patients at different functional conditions between any BIA parameters and FT3 serum values, except for the patient with myxedema, that showed a picture similar to that seen in COVID-19 patients with NTIS. Since the Na+/K+ pump is a well-known T3 target, we measured the mRNA expression levels of the two genes coding for the two major isoforms of this pump. We demonstrated that COVID-19 patients with NTIS had lower levels of mRNA of both genes in the peripheral blood mononuclear cells (PBMC)s obtained from our patients during the acute phase of the disease. In addition, we retrieved data from transcriptome analysis, performed on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM)s treated with T3 and we demonstrated that in these cells T3 is able to stimulate the expression of these two genes in a dose-dependent manner. CONCLUSIONS: In conclusion, we demonstrated that measurement of BIA parameters is a useful method to analyze water and salt retention in COVID-19 patients hospitalized in ICU and, in particular, in those that develop NTIS. Our results indicate that NTIS has peculiar similarities with myxedema seen in severe hypothyroid patients, albeit it occurs more rapidly. The Na+/K+ pump is a possible target of T3 action, involved in the pathogenesis of the anasarcatic condition observed in our COVID-19 patients with NTIS. Finally, measurement of BIA parameters may represent good endpoints to evaluate the benefit of future clinical interventional trials, based on the administration of T3 in patients with NTIS.

Galectin-3: One Molecule for an Alphabet of Diseases, from A to Z.

Galectin-3 (Gal-3) regulates basic cellular functions such as cell-cell and cell-matrix interactions, growth, proliferation, differentiation, and inflammation. It is not surprising, therefore, that this protein is involved in the pathogenesis of many relevant human diseases, including cancer, fibrosis, chronic inflammation and scarring affecting many different tissues. The papers published in the literature have progressively increased in number during the last decades, testifying the great interest given to this protein by numerous researchers involved in many different clinical contexts. Considering the crucial role exerted by Gal-3 in many different clinical conditions, Gal-3 is emerging as a new diagnostic, prognostic biomarker and as a new promising therapeutic target. The current review aims to extensively examine the studies published so far on the role of Gal-3 in all the clinical conditions and diseases, listed in alphabetical order, where it was analyzed.

Thyroid hormones regulate cardiac repolarization and QT-interval related gene expression in hiPSC cardiomyocytes.

Prolongation of cardiac repolarization (QT interval) represents a dangerous and potentially life-threatening electrical event affecting the heart. Thyroid hormones (THs) are critical for cardiac development and heart function. However, little is known about THs influence on ventricular repolarization and controversial effects on QT prolongation are reported. Human iPSC-derived cardiomyocytes (hiPSC-CMs) and multielectrode array (MEA) systems were used to investigate the influence of 3,3',5-triiodo-L-Thyronine (T3) and 3,3',5,5'-tetraiodo-L-Thyronine (T4) on corrected Field Potential Duration (FPDc), the in vitro analog of QT interval, and on local extracellular Action Potential Duration (APD). Treatment with high THs doses induces a significant prolongation of both FPDc and APD, with the strongest increase reached after 24 h exposure. Preincubation with reverse T3 (rT3), a specific antagonist for nuclear TH receptor binding, significantly reduces T3 effects on FPDc, suggesting a TRs-mediated transcriptional mechanism. RNA-seq analysis showed significant deregulation in genes involved in cardiac repolarization pathways, including several QT-interval related genes. In conclusion, long-time administration of high THs doses induces FPDc prolongation in hiPSC-CMs probably through the modulation of genes linked to QT-interval regulation. These results open the way to investigate new potential diagnostic biomarkers and specific targeted therapies for cardiac repolarization dysfunctions.

Modulation of the apoptotic pathway in skeletal muscle models: the role of growth hormone.

Despite numerous studies on the role of growth hormone (GH), its function in skeletal muscle apoptosis secondary to various stimuli is poorly understood. In this study, we used rodent muscle cell lines to analyse cell growth and survival as well as the morphological and molecular markers of cell death in C2C12 and L6C5 myoblasts. These cells were treated either in the presence or absence of GH under serum starvation conditions or in the pro-apoptotic concentrations of hydrogen peroxide (H2O2). Although the cells were responsive to the presence of GH, we did not observe GH modulation of cell growth and survival. The presence of GH did not affect the cell death programme or the expression of apoptotic markers in basal conditions or under oxidative stress. In conclusion, this study indicated that GH "by itself" is not effective in modulating the intracellular pathways leading to cell survival or cell death induced by apoptotic stimuli.

Generation and characterization of the human induced pluripotent stem cell (hiPSC) line NCUFi001-A from a patient carrying KCNQ1 G314S mutation.

In this study we describe the generation and characterization of an human induced pluripotent stem cell (hiPSC) line from a long QT syndrome type 1 (LQT1) patient carrying the KCNQ1 c.940 G > A (p.Gly314Ser) mutation. This patient-specific iPSC line has been obtained by using non-integrational Sendai reprogramming method, expresses pluripotency markers and has the capacity to differentiate into the three germ layers and into spontaneously beating cardiomyocytes (iPSC-CMs).

HIPK2 Is Required for Midbody Remnant Removal Through Autophagy-Mediated Degradation.

At the end of abscission, the residual midbody forms the so-called midbody remnant (MBR), a platform affecting cell fate with emerging key role in differentiation, development, and tumorigenicity. Depending on cell type and pathophysiological context, MBRs undergo different outcomes: they can be retained, released, internalized by nearby cells, or removed through autophagy-mediated degradation. Although mechanisms underlying MBR formation, positioning, and processing have been recently identified, their regulation is still largely unknown. Here, we report that the multifunctional kinase HIPK2 regulates MBR processing contributing to MBR removal. In the process of studying the role of HIPK2 in abscission, we observed that, in addition to cytokinesis failure, HIPK2 depletion leads to significant accumulation of MBRs. In particular, we detected comparable accumulation of MBRs after HIPK2 depletion or treatment with the autophagic inhibitor chloroquine. In contrast, single depletion of the two independent HIPK2 abscission targets, extrachromosomal histone H2B and severing enzyme Spastin, only marginally increased MBR retention, suggesting that MBR accumulation is not just linked to cytokinesis failure. We found that HIPK2 depletion leads to (i) increased levels of CEP55, a key effector of both midbody formation and MBR degradation; (ii) decreased levels of the selective autophagy receptors NBR1 and p62/SQSTM1; and (iii) impaired autophagic flux. These data suggest that HIPK2 contributes to MBR processing by regulating its autophagy-mediated degradation.

Telomere length is independently associated with age, oxidative biomarkers, and sport training in skeletal muscle of healthy adult males.

In skeletal muscle, which mainly contains postmitotic myonuclei, it has been suggested that telomere length remains roughly constant throughout adult life, or shortens in response to physiopathological conditions in muscle diseases or in the elderly. However, telomere length results from both the replicative history of a specific tissue and the exposure to environmental, DNA damage-related factors, therefore the predictive biological significance of telomere measures should combine the analysis of the various interactive factors. In the present study, we analysed any relationship between telomere length [mean and minimum terminal restriction fragment (TRF) length] chronological age, oxidative damage (4-HNE, protein carbonyls), catalase activity, and heat shock proteins expression (αB-crystallin, Hsp27, Hsp90) in semitendinous muscle biopsies of 26 healthy adult males between 20 and 50 years of age, also exploring the influence of regular exercise participation. The multiple linear regression analysis identified age, 4-HNE, catalase, and training status as significant independent variables associated with telomere length and jointly accounting for ∼30-36% of interindividual variation in mean and/or minimum TRF length. No association has been identified between telomere length and protein carbonyl, αB-crystallin, Hsp27, and Hsp90, as well as between age and the variables related to stress response. Our results showed that skeletal muscle from healthy adults displays an age-dependent telomere attrition and that oxidised environment plays an age-independent contribution, partially influenced by exercise training.

Regular exercise participation improves genomic stability in diabetic patients: an exploratory study to analyse telomere length and DNA damage.

Physical activity has been demonstrated to be effective in the prevention and treatment of different chronic conditions, including type 2 diabetes (T2D). In particular, several studies highlighted how the beneficial effects of physical activity may be related to the stability of the DNA molecule, such as longer telomeric ends. Here we analyze the effect of exercise training on telomere length, spontaneous and H2O2-induced DNA damage, as well as the apoptosis level in leukocytes from untrained or trained T2D patients vs. age-matched control subjects (CS) (57-66 years). Moreover, expression analysis of selected genes belonging to DNA repair systems, cell cycle control, antioxidant and defence systems was performed. Subjects that participated in a regular exercise program showed a longer telomere sequence than untrained counterparts. Moreover, ex vivo treatment of leukocytes with H2O2 highlighted that: (1) oxidative DNA damage induced similar telomere attrition in all groups; (2) in T2D subjects, physical activity seemed to prevent a significant increase of genomic oxidative DNA damage induced by chronic exposure to pro-oxidant stimulus, and (3) decreased the sensitivity of leukocytes to apoptosis. Finally, the gene expression analysis in T2D subjects suggested an adaptive response to prolonged exercise training that improved the response of specific genes.

CDKL5 localizes at the centrosome and midbody and is required for faithful cell division.

The cyclin-dependent kinase-like 5 (CDKL5) gene has been associated with rare neurodevelopmental disorders characterized by the early onset of seizures and intellectual disability. The CDKL5 protein is widely expressed in most tissues and cells with both nuclear and cytoplasmic localization. In post-mitotic neurons CDKL5 is mainly involved in dendritic arborization, axon outgrowth, and spine formation while in proliferating cells its function is still largely unknown. Here, we report that CDKL5 localizes at the centrosome and at the midbody in proliferating cells. Acute inactivation of CDKL5 by RNA interference (RNAi) leads to multipolar spindle formation, cytokinesis failure and centrosome accumulation. At the molecular level, we observed that, among the several midbody components we analyzed, midbodies of CDKL5-depleted cells were devoid of HIPK2 and its cytokinesis target, the extrachromosomal histone H2B phosphorylated at S14. Of relevance, expression of the phosphomimetic mutant H2B-S14D, which is capable of overcoming cytokinesis failure in HIPK2-defective cells, was sufficient to rescue spindle multipolarity in CDKL5-depleted cells. Taken together, these results highlight a hitherto unknown role of CDKL5 in regulating faithful cell division by guaranteeing proper HIPK2/H2B functions at the midbody.

Comparative analysis of diagnostic performance, feasibility and cost of different test-methods for thyroid nodules with indeterminate cytology.

Since it is impossible to recognize malignancy at fine needle aspiration (FNA) cytology in indeterminate thyroid nodules, surgery is recommended for all of them. However, cancer rate at final histology is <30%. Many different test-methods have been proposed to increase diagnostic accuracy in such lesions, including Galectin-3-ICC (GAL-3-ICC), BRAF mutation analysis (BRAF), Gene Expression Classifier (GEC) alone and GEC+BRAF, mutation/fusion (M/F) panel, alone, M/F panel+miRNA GEC, and M/F panel by next generation sequencing (NGS), FDG-PET/CT, MIBI-Scan and TSHR mRNA blood assay.We performed systematic reviews and meta-analyses to compare their features, feasibility, diagnostic performance and cost. GEC, GEC+BRAF, M/F panel+miRNA GEC and M/F panel by NGS were the best in ruling-out malignancy (sensitivity = 90%, 89%, 89% and 90% respectively). BRAF and M/F panel alone and by NGS were the best in ruling-in malignancy (specificity = 100%, 93% and 93%). The M/F by NGS showed the highest accuracy (92%) and BRAF the highest diagnostic odds ratio (DOR) (247). GAL-3-ICC performed well as rule-out (sensitivity = 83%) and rule-in test (specificity = 85%), with good accuracy (84%) and high DOR (27) and is one of the cheapest (113 USD) and easiest one to be performed in different clinical settings.In conclusion, the more accurate molecular-based test-methods are still expensive and restricted to few, highly specialized and centralized laboratories. GAL-3-ICC, although limited by some false negatives, represents the most suitable screening test-method to be applied on a large-scale basis in the diagnostic algorithm of indeterminate thyroid lesions.

Combined clinical and ultrasound follow-up assists in malignancy detection in Galectin-3 negative Thy-3 thyroid nodules.

The use of galectin-3 ThyroTest in the preoperative evaluation of cytologically indeterminate (Thy-3) thyroid nodules has been largely validated by retrospective and prospective multicentre studies. Here we report the results of galectin-3 ThyroTest routinely applied in the management of Thy-3 nodules in combination with clinical and ultrasonography (US) examination, in which galectin-3 positive nodules were directly referred to surgery whereas galectin-3 negative lesions were considered for clinical and US long-term follow-up. A cohort of 331 patients, bearing 340 thyroid Thy-3 nodules, was enrolled and subjected to galectin-3 expression analysis. A total of 256 galectin-3 negative nodules were directed to periodical clinical and US examination, while 84 galectin-3 positive cases were referred to surgery. Excluding 63 dropout patients plus 15 patients that were operated because of clinical reasons the remaining 176 galectin-3 negative nodules were followed with clinical and US examination for an average period of 31 months. During the follow-up, the volume of galectin-3 negative nodules was unchanged in 85 cases (48 %), reduced in 47 (27 %), and increased in 44 (25 %). Based on combined clinical features and US follow-up results, a total of 36 out of 191 galectin-3 negative nodules (19 %) were referred to surgery, with a final histological finding of 28 benign lesions, three follicular tumor of uncertain malignant potential (FT-UMP), and five malignant lesions, corresponding to a 7 % false negative rate. In the group of 84 galectin-3 positive nodules, we detected 65 thyroid cancers with a prevalence of 77 %, 12 FT-UMPs, and 7 false positive lesions, corresponding to a 4 % false positive rate. A total of 150 patients were not operated and are still under clinical and US monitoring while surgery was performed in 118 patients with a final 70 thyroid cancers diagnosed, corresponding to a 59 % prevalence of malignancy detected at surgery and to a 26 % prevalence of malignancy among the entire Thy-3 nodule population. Galectin-3 ThyroTest is an easy and cheap diagnostic procedure that integrates conventional fine-needle-aspiration cytology, reduces the number of unnecessary thyroidectomies and increases the rate of malignancy at surgery. Clinical and US follow-up of galectin-3 negative lesions allows to further reduce false negative cases.

Acute exercise modulates BDNF and pro-BDNF protein content in immune cells.

PURPOSE: Although several studies have shown that immune cells stimulated by in vitro stress are capable to produce neurotrophins, there is still no evidence whether physiological stress, such as exercise, can modulate the in vivo levels of brain-derived neurotrophic factor (BDNF) in peripheral blood mononuclear cells (PBMCs). METHODS: This work investigated whether acute exercise modulates the expression of BDNF, pro-BDNF, and p75(NTR) in the PBMCs of 10 healthy young men who performed a cycling incremental test to exhaustion (MAX) or exercised at individual anaerobic threshold (IAT). The PBMC expression of stress response proteins and the level of circulating BDNF, vascular endothelial growth growth factor, platelet-derived growth factor subunit B, basic fibroblast growth factor pro-inflammatory, and anti-inflammatory cytokines were analyzed as well. RESULTS: A major finding is that both sessions of acute exercise regulated the content of BDNF isoforms within PBMCs in a manner related to the physiological stress exerted. Although the pro-BDNF increased after both MAX and IAT protocols, BDNF showed a kinetics dependent on exercise type: MAX induced a 54% protein increase immediately after exercise, followed by a significant drop 60 min after its conclusion (38% lower than the baseline). Differently, in the IAT, BDNF increased significantly up to 75% from the baseline throughout the recovery phase. All physiological parameters, as well as the p75(NTR) receptor and the stress-inducible proteins, were also differently regulated by the two exercise conditions. CONCLUSIONS: These data supported the hypothesis that PBMCs might produce and secrete BDNF isoforms, as well as modulate the proteins p75(NTR) , Bcl-xL, hsp90, hsp27, and αB-crystallin, as part of the physiological stress response induced by acute exercise, offering a novel example of bidirectional interaction between nervous and immune systems.

NF-Y dependent epigenetic modifications discriminate between proliferating and postmitotic tissue.

The regulation of gene transcription requires posttranslational modifications of histones that, in concert with chromatin remodeling factors, shape the structure of chromatin. It is currently under intense investigation how this structure is modulated, in particular in the context of proliferation and differentiation. Compelling evidence suggests that the transcription factor NF-Y acts as a master regulator of cell cycle progression, activating the transcription of many cell cycle regulatory genes. However, the underlying molecular mechanisms are not yet completely understood. Here we show that NF-Y exerts its effect on transcription through the modulation of the histone "code". NF-Y colocalizes with nascent RNA, while RNA polymerase II is I phosphorylated on serine 2 of the YSPTSPS repeats within its carboxyterminal domain and histones are carrying modifications that represent activation signals of gene expression (H3K9ac and PAN-H4ac). Comparing postmitotic muscle tissue from normal mice and proliferating muscles from mdx mice, we demonstrate by chromatin immunoprecipitation (ChIP) that NF-Y DNA binding activity correlates with the accumulation of acetylated histones H3 and H4 on promoters of key cell cycle regulatory genes, and with their active transcription. Accordingly, p300 is recruited onto the chromatin of NF-Y target genes in a NF-Y-dependent manner, as demonstrated by Re-ChIP. Conversely, the loss of NF-Y binding correlates with a decrease of acetylated histones, the recruitment of HDAC1, and a repressed heterochromatic state with enrichment of histones carrying modifications known to mediate silencing of gene expression (H3K9me3, H3K27me2 and H4K20me3). As a consequence, NF-Y target genes are downregulated in this context. In conclusion, our data indicate a role of NF-Y in modulating the structure and transcriptional competence of chromatin in vivo and support a model in which NF-Y-dependent histone "code" changes contribute to the proper discrimination between proliferating and postmitotic cells in vivo and in vitro.