Effects of P53 mutants on the deregulation of P53-MDM2 feedback loop

Aim: P53 and MDM2 constitute a negative feedback loop. The coexistence of these two molecules in sarcomas and lung carcinomas has been associated with poor prognosis. In the present PhD study the status of p53 and MDM2 was evaluated in a series of lung carcinomas, and the role of p53 mutants was examined, especially, in the cases where a deregulation of the above mentioned feedback loop was found. Material and Methods: The sample comprised 21 squamous cell carcinomas, 19 adenocarcinomas and 10 small cell carcinomas of the lung. Evaluation of p53 and MDM2 protein status was based on immunohistochemical and Western blot analysis. Moreover, p53 status was examined by mutation analysis (SSCP and Sequencing) and LOH analysis (using Southern blot), whereas MDM2 status was focused on mRNA levels (Northern blot) and gene-amplification (differential PCR). Next, the role of certain p53 mutants, derived from specimens characterized by deregulation in the p53-MDM2 feedback loop, was examined. In detail, certain p53 mutants were examined for their ability i) to bind the p53 responsive element (RE)of the MDM2 gene (Retardation assay) and ii) to transactivate the luciferase reference gene via MDM2-p53RE (side-directed mutagenesis and luciferase assay after transfection of H1299 p53-null cell line). Results: P53 immunopositivity was observed in 28 cases (56%). This was associated with p53 mutation (24 of 28 specimens) and loss of heterozygosity (13 of 16 informative cases) (p<0.0001 και p=0.015, respectively). Protein analysis for MDM2 revealed nuclear immunostaining in 30 (60%) samples, and the existence of three isoforms: p90, p76/74 and p57. The MDM2 overexpression was accompanied by increased mRNA levels and not by gene amplification. Co-existence of p53 and MDM2 was observed in 21 of 28 cases with mutant p53. The results of the binding assay for 12 selected p53 mutants were in accordance with those of the protein analysis. The luciferase assay experiments showed the importance of certain mutations for special biological properties of the p53. Conclusions: Concurrent nuclear immunostaining of p53 and MDM2 was observed in a subset of lung carcinomas. This phenomenon, that was associated with poor differentiation and lymph node invasion, was -in part- a result of the function of special p53 mutants possessing oncogenic properties. In general, it was found that the role of the p53 mutants was depended on the position and the nature of the mutation and the presence of the wild type p53.Σκοπός: Τα γονίδια TP53 και MDM2 αλληλεπιδρούν σε ένα παλίνδρομο αρνητικό μηχανισμό ανάδρασης. Έχει διαπιστωθεί ότι συνύπαρξη των p53 και MDM2 σε σαρκώματα και σε νεοπλάσματα πνεύμονα συνοδεύεται από χειρότερη πρόγνωση. Στην παρούσα διδακτορική διατριβή μελετήθηκαν τα γονίδια p53 και MDM2 στον καρκίνο του πνεύμονα, και διερευνήθηκε ο ρόλος των μεταλλαγμένων p53 στις περιπτώσεις που παρατηρείται διαταραχή του προαναφερόμενου μηχανισμού ανάδρασης. Υλικό και Μέθοδοι: Το υλικό αποτελείται από 21 επιδερμοειδή καρκινώματα, 19 αδενοκαρκινώματα και 10 μικροκυτταρικά καρκινώματα του πνεύμονα. Η εκτίμηση των πρωτεϊνών p53 και MDM2 έγινε σε κυτταρικό- ιστικό επίπεδο με τη χρήση της ανοσοϊστοχημείας και σε μοριακό επίπεδο με τη μέθοδο Western. Περαιτέρω, η έκφρασή τους συνδυάστηκε με τη μοριακή ταυτότητα του γονιδίου τους που αλλοιώνει το φυσιολογικό τους ρόλο. Συγκεκριμένα, η έκφραση της p53 συσχετίστηκε με την κατάσταση του γονιδίου TP53 [εξέταση μεταλλαγών με τις μέθοδους SSCP και Sequencing και ελλείψεως γονιδίου (έλεγχος απώλειας ετεροζυγωτίας) με τη μέθοδο Southern], ενώ η έκφραση της MDM2 συνδυάστηκε με τη μεταγραφική ενεργοποίηση του γονιδίου [έλεγχος επίπεδων μεταγράφων με τη μέθοδο Northern] και την ύπαρξη γονιδιακής ενίσχυσης [με τη μέθοδο του διαφορικού-PCR]. Στη συνέχεια, σε επιλεγμένα δείγματα που διαπιστώθηκε διαταραχή του αρνητικού μηχανισμού ανάδρασης p53-MDM2 εξετάστηκε ο ρόλος των μεταλλαγμένων μορφών της p53. Συγκεκριμένα, μελετήθηκε η ικανότητα των μεταλλαγμένων p53 αφενός να προσδένονται στα ειδικά στοιχεία ανταπόκρισης για το p53 της ρυθμιστικής περιοχής του γονιδίου MDM2 (με τη μέθοδο EMSA) και αφετέρου η ικανότητά συγκεκριμένων από αυτά (κατασκευή μεταλλαγμένων p53 με τη μέθοδο της κατευθυνόμενης μεταλλαξογένεσης) για μεταγραφική ενεργοποίηση γονιδίου αναφοράς (λουσιφεράση) υπό τη ρύθμιση του συγκεκριμένου στοιχείου ανταπόκρισης (πειράματα διαμόλυνσης με γονίδιο αναφοράς αυτό της λουσιφεράσης). Αποτελέσματα: Ανοσοϊστοχημική εντόπιση της p53 διαπιστώθηκε σε 28 περιστατικά (56%). Από αυτά βρέθηκε μετάλλαξη του γονιδίου σε 24 περιπτώσεις (86%) η οποία συνοδευόταν από απώλεια ετεροζυγωτίας σε 13 από τα 16 αξιολογίσιμα δείγματα (81%) (p<0.0001 και p=0.015, αντίστοιχα). Ανοσοϊστοχημική εντόπιση της MDM2 παρατηρήθηκε σε 30 περιστατικά (60%), ενώ ανευρέθησαν τρείς πρωτεϊνικές ισομορφές: p90, p57 και p76/74. Τα αυξημένα επίπεδα της MDM2 οφείλονται σε αυξημένα επίπεδα mRNA τα οποία δεν σχετίζονται με γονιδιακή ενίσχυση. Ταυτόχρονη ανοσοϊστοχημική εντόπιση των p53 και MDM2 διαπιστώθηκε σε 21 από τα 28 περιστατικά με μεταλλαγμένη την p53 (p=0.0004). Τα αποτελέσματα της εξέτασης της δεσμευτικής ικανότητας στο στοιχείο ανταπόκρισης για το p53 του MDM2 δώδεκα επιλεγμένων μεταλλαγμένων p53 ήταν σε συμφωνία με τα παρατηρούμενα πρωτεϊνικά επίπεδα της MDM2. Τα πειράματα διαμόλυνσης με 4 κατασκευασμένα μεταλλαγμένα μόρια p53 έδειξαν τη σημασία τόσο της θέσης όσο και της φύσης των μεταλλάξεων για τη λειτουργικότητα του μορίου. Συμπεράσματα: Τουλάχιστον, σε μια ομάδα καρκινωμάτων του πνεύμονα διαπιστώθηκε ταυτόχρονη έκφραση των πρωτεϊνών p53 και MDM2. Το φαινόμενο αυτό, που συνδυάστηκε με χαμηλό βαθμό διαφοροποίησης και λεμφαδενικές μεταστάσεις, εν μέρει δικαιολογήθηκε με τη δράση συγκεκριμένων μεταλλαγμένων p53 πρωτεϊνών που παρουσιάζουν ογκογόνες ιδιότητες. Γενικότερα παρατηρήθηκε ότι ο ρόλος των μεταλλάξεων σχετίζεται με το είδος και τη θέση τους καθώς και με τη συνύπαρξη φυσιολογικής p5

Low back pain induces disability of women in primary uncomplicated pregnancy

Study design: A consecutive case series study Purpose: To investigate whether Low Back Pain (LBP) in women with primary singleton pregnancy induces disability. Background: LBP is reported to be increased in pregnants than in non-pregnant women. Different outcome measures have been used to search for correlations between pain and disability. Methods: 167 pregnant women aged 30 &plusmn; 3.5 years participated. Two equal categorial age groups were constructed: Group A included women aged 23 - 29 years, and Group B women aged 30-39 years. Their weight was 76 &plusmn; 13 kg prepartum and the Body Mass index (BMI) was 28 &plusmn; 4 prepartum. Visual Analogue Scale (VAS) was used for LBP pain intensity and Oswestry Disability Scale (ODI) for disability estimation in the last three months prepartum and in the first three months postpartum. Results: The women weight was 67 &plusmn; 13 kg postpartum. The BMI was 24 &plusmn; 4 postpartum. There was no difference in VAS and ODI scores versus BMI, weight and height between the two age groups in both periods of observation: prepartum and postpartum. Prepartum, 81.4% of women claimed LBP that dropped to 55.5% postpartum. ODI score dropped from 19.5 &plusmn; 13.6% prepartum to 11 &plusmn; 12% postpartum. The ODI subscales that showed significant reduction postpartum were: Pain intensity (P = 0.002); working (P = 0.009); sitting (P = 0.004); standing (P = 0.003); sleeping (P = 0.008); and traveling (P = 0.006). VAS prepartum was increasing as the weight was increasing in both periods of observation (P = 0.015 and P=0.051) respectively. VAS prepartum was significantly correlated with BMI prepartum (P = 0.019) and postpartum (P = 0.028). Discussion: Physical disability in pregnant women was low and reduced following delivery. Disability was linked with LBP intensity, weight, BMI and height, but not with age or educational level.&nbsp

Single Step Laser Transfer and Laser Curing of Ag NanoWires: A Digital Process for the Fabrication of Flexible and Transparent Microelectrodes

Ag nanowire (NW) networks have exquisite optical and electrical properties which make them ideal candidate materials for flexible transparent conductive electrodes. Despite the compatibility of Ag NW networks with laser processing, few demonstrations of laser fabricated Ag NW based components currently exist. In this work, we report on a novel single step laser transferring and laser curing process of micrometer sized pixels of Ag NW networks on flexible substrates. This process relies on the selective laser heating of the Ag NWs induced by the laser pulse energy and the subsequent localized melting of the polymeric substrate. We demonstrate that a single laser pulse can induce both transfer and curing of the Ag NW network. The feasibility of the process is confirmed experimentally and validated by Finite Element Analysis simulations, which indicate that selective heating is carried out within a submicron-sized heat affected zone. The resulting structures can be utilized as fully functional flexible transparent electrodes with figures of merit even higher than 100. Low sheet resistance (&lt;50 Ohm/sq) and high visible light transparency (&gt;90%) make the reported process highly desirable for a variety of applications, including selective heating or annealing of nanocomposite materials and laser processing of nanostructured materials on a large variety of optically transparent substrates, such as Polydimethylsiloxane (PDMS)

The role of Src & ERK1/2 kinases in inspiratory resistive breathing induced acute lung injury and inflammation

Abstract Background Inspiratory resistive breathing (IRB), a hallmark of obstructive airway diseases, is associated with large negative intrathoracic pressures, due to strenuous contractions of the inspiratory muscles. IRB is shown to induce lung injury in previously healthy animals. Src is a multifunctional kinase that is activated in the lung by mechanical stress. ERK1/2 kinase is a downstream target of Src. We hypothesized that Src is activated in the lung during IRB, mediates ERK1/2 activation and IRB-induced lung injury. Methods Anaesthetized, tracheostomized adult rats breathed spontaneously through a 2-way non-rebreathing valve. Resistance was added to the inspiratory port to provide a peak tidal inspiratory pressure of 50% of maximum (inspiratory resistive breathing). Activation of Src and ERK1/2 in the lung was estimated during IRB. Following 6 h of IRB, respiratory system mechanics were measured by the forced oscillation technique and bronchoalveolar lavage (BAL) was performed to measure total and differential cell count and total protein levels. IL-1b and MIP-2a protein levels were measured in lung tissue samples. Wet lung weight to total body weight was measured and Evans blue dye extravasation was estimated to measure lung permeability. Lung injury was evaluated by histology. The Src inhibitor, PP-2 or the inhibitor of ERK1/2 activation, PD98059 was administrated 30 min prior to IRB. Results Src kinase was activated 30 min after the initiation of IRB. Src inhibition ameliorated the increase in BAL cellularity after 6 h IRB, but not the increase of IL-1β and MIP-2a in the lung. The increase in BAL total protein and lung injury score were not affected. The increase in tissue elasticity was partly inhibited. Src inhibition blocked ERK1/2 activation at 3 but not at 6 h of IRB. ERK1/2 inhibition ameliorated the increase in BAL cellularity after 6 h of IRB, blocked the increase of IL-1β and returned Evans blue extravasation and wet lung weight to control values. BAL total protein and the increase in elasticity were partially affected. ERK1/2 inhibition did not significantly change total lung injury score compared to 6 h IRB. Conclusions Src and ERK1/2 are activated in the lung following IRB and participate in IRB-induced lung injury

Low T3 Syndrome in severely ill patients with COVID-19 infection

Introduction The coronavirus disease (COVID-19) is an infectious disease, caused by the SARS-CoV-2 virus, which causes severe respiratory disease. Critical ill patients often experience a condition known as Low T3 Syndrome (LT3S). Previous studies showed an association between low FT3 levels and mortality among patients with COVID-19. Moreover, thyroid hormones might be altered by cigarette consumption. Τhe aim of this study was to investigate the association of LT3S with mortality and the severity and risk of intubation in critically ill patients with COVID-19 infection, and to explore whether this association is confounded by smoking. Methods A total of 105 critically ill patients aged ≥18 years, with laboratoryconfirmed (RT-PCR) COVID-19 were enrolled. The study was conducted between January 2021 and October 2021 in the Intensive Care Unit of the 1st Department Respiratory Medicine in ‘Sotiria’ Hospital and laboratory data and clinical information were retrieved retrospectively from the electronic patients record. LT3S was defined as serum levels of FT3 <2.3 pg/mL with low or normal TSH levels. Patients were divided into two groups according to serum FT3 values: group with LT3S and group without LT3S. Mortality in the ICU was the primary outcome of the study, while the risk of intubation was a secondary outcome. Results In all, 43 out of the 105 included patients were diagnosed with LT3S. Patients in the LT3S group were older than those with non LT3S [median (IQR): 62 (13.7) vs 52.8 (15.5), p=0.011]. Non-statistically significantly higher mortality rate, SOFA and APACHE II scores were observed in the LT3S group compared to no LT3S group (p=0.080, p=0.311 and p=0.079, respectively). Moreover, LT3S was not associated with high risk of intubation (HR=1.32; 95% CI: 0.78–2.22). Twenty-five patients (58.1%) in the LT3S group were never smokers, versus 41 (66.1%) patients in the non LT3S group. Never smokers with LT3S had significantly higher mortality rate than never smokers without LT3S (40% vs 17.1%, p=0.039), and LT3S in the never smoking subgroup was associated with an increased risk of intubation (HR=2.21; 95% CI: 1.18–4.16). Conclusions LT3S was found to be associated with mortality of patients with critical COVID-19 among never smokers but not among ex-smokers or active smokers. This finding may denote that smoking may act as a confounder of the association between LT3S and mortality. Further investigation is needed to demonstrate the impact of LT3S in critically ill patients with COVID-19 infection, as well as the role of smoking in the development of the syndrome

Nitric Oxide Stimulates Interleukin-6 Production in Skeletal Myotubes

Strenuous exercise leads to the up-regulation of interleukin-6 (IL-6) production and enhanced nitric oxide (NO) release within the contracting skeletal muscles. In this study, we investigated whether NO regulates IL-6 production in C2C12 myotubes. These cells exhibited a concentration-dependent increase in IL-6 production upon stimulation with NO donors (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diola te (DETA-NONOate), (Z)-1-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate (PAPA-NONOate), and sodium nitroprusside (SNP). This treatment did not alter cGMP levels nor did the soluble guanylyl cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one(ODQ), alter this response. The NO-independent sGC activator 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl] -pyrimidin-4-ylamine (BAY41-2272) and cyclic guanosine monophosphate (cGMP) analog 8Br-cGMP failed to induce IL-6 production. Upon exposure to NO donors, we observed an increase in Erk1/2 and p38 MAPK phosphorylation but not in SAPK/JNK. In addition, NO-induced IL-6 release was inhibited in a concentration-dependent fashion by the MEK1/2 inhibitor PD98059 and the p38 MAPK inhibitor SB203580 but not by the SAPK/JNK inhibitor SP600125. We conclude that NO-stimulated IL-6 production in differentiated C2C12 myotubes is cGMP-independent and mediated by activation of MAPK pathways

Asymmetric evolution of anterior chest wall blood supply in female adolescents with progressive right-convex thoracic idiopathic scoliosis

Breast asymmetry was believed to be related to asymmetry of anterior chest wall blood supply and subsequently to aetiology of idiopathic thoracic scoliosis in female adolescents. Recent investigations on the anterior chest wall blood supply with Colour Doppler Ultrasonography (CDU) in such individuals did not show anatomical and hemodynamic abnormalities. The present study investigated the evolution of anterior chest wall blood supply in these individuals over a 2-year period. Twenty female adolescents with progressive right-convex idiopathic thoracic scoliosis (scoliotics), who were during the study in therapy with horacolumbosacral orthosis (TLSO) and 20 age-matched girls, without spine deformity (controls) were studied with CDU [internal mammary artery (IMA)] twice within the 2-year period. IMA-anatomical parameters [lumen diameter (D) and cross sectional area (AR)] as well as hemodynamic flow parameters [time average mean flow velocity and flow volume per minute (FV)] were measured. In the 2-year-period of observation, TLSO prevented scoliosis progression (P = 0.004), while IMA-AR decreased bilaterally in the individuals of both groups (P < 0.03). In the last evaluation: in scoliotics right IMA FV decreased (P < 0.04), while in controls IMA FV decreased bilaterally (P < 0.03); left IMA FV was significantly higher (P < 0.05) in scoliotics than in controls. The significant, within the 2-year period, decrease of IMA-diameter, cross-sectional area, and flow volume seems to be a physiological ageing process because it was observed in all individuals (scoliotics and controls), and thus these anatomic and hemodynamic changes seem not to have been affected by bracing. The maintenance of left flow volume of IMA in the pre-brace levels in scoliotics was the most significant finding of this investigation. In conclusion, this study provided evidence for abnormalities in the evolution of anterior chest wall blood supply in female adolescents with progressive right-convex female thoracic scoliosis. Further studies are needed to investigate if this asymmetric blood evolution contributes to the development of this pattern of scoliosis in girls