Immunohistochemical analysis of adhesive papillae of Clavelina lepadiformis (Müller, 1776) and Clavelina phlegraea (Salfi, 1929) (Tunicata, Ascidiacea)

Almost all ascidian larvae bear three mucus secreting and sensory organs, the adhesive papillae, at the anterior end of the trunk, which play an important role during the settlement phase. The morphology and the cellular composition of these organs varies greatly in the different species. The larvae of the Clavelina genus bear simple bulbous papillae, which are considered to have only a secretory function. We analysed the adhesive papillae of two species belonging to this genus, C. lepadiformis and C. phlegraea, by histological sections and by immunolocalisation of β-tubulin and serotonin, in order to better clarify the cellular composition of these organs. We demonstrated that they contain at least two types of neurons: central neurons, bearing microvilli, and peripheral ciliated neurons. Peripheral neurons of C. lepadiformis contain serotonin. We suggest that these two neurons play different roles during settlement: the central ones may be chemo- or mechanoreceptors that sense the substratum, and the peripheral ones may be involved in the mechanism that triggers metamorphosis

Machine learning predicts lung recruitment in acute respiratory distress syndrome using single lung CT scan

Background: To develop and validate classifier models that could be used to identify patients with a high percentage of potentially recruitable lung from readily available clinical data and from single CT scan quantitative analysis at intensive care unit admission. 221 retrospectively enrolled mechanically ventilated, sedated and paralyzed patients with acute respiratory distress syndrome (ARDS) underwent a PEEP trial at 5 and 15 cmH2O of PEEP and two lung CT scans performed at 5 and 45 cmH2O of airway pressure. Lung recruitability was defined at first as percent change in not aerated tissue between 5 and 45 cmH2O (radiologically defined; recruiters: Δ45-5non-aerated tissue > 15%) and secondly as change in PaO2 between 5 and 15 cmH2O (gas exchange-defined; recruiters: Δ15-5PaO2 > 24 mmHg). Four machine learning (ML) algorithms were evaluated as classifiers of radiologically defined and gas exchange-defined lung recruiters using different models including different variables, separately or combined, of lung mechanics, gas exchange and CT data. Results: ML algorithms based on CT scan data at 5 cmH2O classified radiologically defined lung recruiters with similar AUC as ML based on the combination of lung mechanics, gas exchange and CT data. ML algorithm based on CT scan data classified gas exchange-defined lung recruiters with the highest AUC. Conclusions: ML based on a single CT data at 5 cmH2O represented an easy-to-apply tool to classify ARDS patients in recruiters and non-recruiters according to both radiologically defined and gas exchange-defined lung recruitment within the first 48 h from the start of mechanical ventilation

Pathophysiology of hypoxemia in mechanically-ventilated patients with COVID-19: A computed tomography study

The pathogenesis of hypoxemia during acute respiratory distress syndrome caused by SARS-CoV-2 infection (C-ARDS) is debated. Some observations led to hypothesize ventilation to perfusion mismatch, rather than anatomical shunt, as the main determinant of hypoxemia. In this observational study 24 C-ARDS patients were studied 1 (0–1) days after intubation. Patients underwent a CT scan analysis to estimate anatomical shunt and a clinical test to measure venous admixture at two fractions of inspired oxygen (FiO2), to eliminate oxygen-responsive mechanisms of hypoxemia (ventilation to perfusion mismatch and diffusion limitation). In 10 out of 24 patients venous admixture was higher than anatomical shunt both at clinical (≈50 %) and 100 % FiO2. These patients were ventilated with a higher PEEP and had lower amount of anatomical shunt compared with patients with venous admixture equal/lower than anatomical shunt. In a subset of C-ARDS patients early after endotracheal intubation, hypoxemia might be explained by an abnormally high perfusion of a relatively low anatomical shunt

Effects of bisphenol A on early development of the ascidian Phallusia mammillata (Chordata, Tunicata)

Bisphenol A (BPA) is an organic compound used in the manufacture of polycarbonate plastic and epoxy resins that is released into the environment from sewage treatment effluent, landfill leachate and degradation of plastic products. BPA can act both as a teratogenic substance and as an endocrine disruptor. The phylogenetic position of tunicates as sister group of vertebrates and their cosmopolitan distribution in marine ecosystems coupled with their ecology and easy manipulability make them reliable model organisms for ecotoxicology bioassays. Here we evaluated the effects of different concentration of BPA (0.1, 0.5, 1, 5, 10, and 20 \ub5M) on ontogenetic processes sensitive to environmental pollutants in the ascidian Phallusia mammillata. To test the effects of this substance on sperm viability we fertilized eggs with sperm pre-exposed for 30 minutes to BPA. One hour post fertilization (hpf) we calculated the percentage of eggs that reached the 2-cell stage compared to control. Then we analyzed the consequences of a fertilization performed directly in BPA solutions, counting the percentage of 2 cell-stage embryos 1 hpf. To test the effects on embryogenesis, we analyzed hatched larvae developed from 2-cell embryos exposed to BPA. We evaluated the number and type of malformations under a dissection microscope and we performed immunostaining of central nervous system (CNS). Exposure of sperm to BPA did not influence fertilization rate. Coexposure of eggs and sperm at concentrations higher than 5 \ub5M caused incomplete division of eggs, producing heart shaped embryos that did not develop further. Regarding embryogenesis, 10 \ub5M BPA caused specific malformations at central nervous system, with an increased distance between otolith and ocellus, or an extrusion of ocellus from the sensory vesicle. Embryonic development was ignificantly altered at 20 \ub5M concentration. 97% of the larvae presented a severely affected phenotype with short and kinked tail. Immunostainig with anti \u3b2-tubulin antibody showed an altered pattern of fibers in CNS. These results showed that the most sensitive process to BPA is the first cell division. When the 2-cell stage embryos are treated, higher concentration are required to alter the correct development of CNS. BPA confirmed its teratogenic effects on ascidians and its interference with CNS development even if the mechanism of action in this group is still to be clarified

Teratogenic Potential of Traditionally Formulated and Nano-Encapsulated Vitamin A in Two Vertebrate Models, Rattus norvegicus and Xenopus Laevis

Nano-encapsulation is applied for the preparation of functional food to preserve micronutrients degradation and to ameliorate their absorption. Being nano-encapsulation already related to increased vitamin A embryotoxicity, we aimed to evaluate the effect of traditionally formulated (BULK-A) and nano-encapsulated vitamin A (NANO-A) in two different vertebrate models: rat post implantation Whole Embryo Culture (WEC) and Frog Embryo Teratogenesis Assay-Xenopus (FETAX). After benchmark-dose modelling, WEC results showed that NANO-A was 7 times more effective than BULK-A, while FETAX results indicated that X. laevis development was affected only by NANO-A. The relative potency of WEC was 14 times the potency of FETAX, suggesting a minor role of preformed vitamin A in X. laevis development in respect to mammal embryogenesis. Results from this work prompt the necessity to monitor the use of food supplemented with NANO A, since even low doses can elicit teratogenic effects on vertebrate embryos due to its increased bioavailability

Lung response to prone positioning in mechanically-ventilated patients with COVID-19

Background: Prone positioning improves survival in moderate-to-severe acute respiratory distress syndrome (ARDS) unrelated to the novel coronavirus disease (COVID-19). This benefit is probably mediated by a decrease in alveolar collapse and hyperinflation and a more homogeneous distribution of lung aeration, with fewer harms from mechanical ventilation. In this preliminary physiological study we aimed to verify whether prone positioning causes analogue changes in lung aeration in COVID-19. A positive result would support prone positioning even in this other population. Methods: Fifteen mechanically-ventilated patients with COVID-19 underwent a lung computed tomography in the supine and prone position with a constant positive end-expiratory pressure (PEEP) within three days of endotracheal intubation. Using quantitative analysis, we measured the volume of the non-aerated, poorly-aerated, well-aerated, and over-aerated compartments and the gas-to-tissue ratio of the ten vertical levels of the lung. In addition, we expressed the heterogeneity of lung aeration with the standardized median absolute deviation of the ten vertical gas-to-tissue ratios, with lower values indicating less heterogeneity. Results: By the time of the study, PEEP was 12 (10–14) cmH2O and the PaO2:FiO2 107 (84–173) mmHg in the supine position. With prone positioning, the volume of the non-aerated compartment decreased by 82 (26–147) ml, of the poorly-aerated compartment increased by 82 (53–174) ml, of the normally-aerated compartment did not significantly change, and of the over-aerated compartment decreased by 28 (11–186) ml. In eight (53%) patients, the volume of the over-aerated compartment decreased more than the volume of the non-aerated compartment. The gas-to-tissue ratio of the ten vertical levels of the lung decreased by 0.34 (0.25–0.49) ml/g per level in the supine position and by 0.03 (− 0.11 to 0.14) ml/g in the prone position (p < 0.001). The standardized median absolute deviation of the gas-to-tissue ratios of those ten levels decreased in all patients, from 0.55 (0.50–0.71) to 0.20 (0.14–0.27) (p < 0.001). Conclusions: In fifteen patients with COVID-19, prone positioning decreased alveolar collapse, hyperinflation, and homogenized lung aeration. A similar response has been observed in other ARDS, where prone positioning improves outcome. Therefore, our data provide a pathophysiological rationale to support prone positioning even in COVID-19

Studio degli effetti miscela sullo sviluppo embrionale in vitro di due composti con diverso meccanismo d’azione (fluconazolo ed etanolo) utilizzando due diversi modelli sperimentali: coltura di embrioni di ratto e esposizione di embrioni di Ascidia

Scopo di questo lavoro \ue8 stato quello di valutare in vitro gli effetti miscela sul potenziale teratogeno della co-esposizione a Fluconazolo (FLUCO, fungicida azolico ampiamente utilizzato in ambito clinico) ed a concentrazioni sub-teratogene di Etanolo (Eth). Nella prima parte di questo studio, embrioni post-impianto di ratto sono stati esposti in vitro a concentrazioni crescenti di FLUCO (62.5-125-250-500\ub5M), alla concentrazione non effetto di Eth (1\ub5L/mL) o sono stati co-esposti a FLUCO 62.5-125-250-500\ub5M ed Eth (1\ub5L/mL). Al termine del periodo di coltura (48 ore) gli embrioni sono stati esaminati morfologicamente e processati per valutare l\u2019espressione dei geni specificamente coinvolti nel metabolismo dell\u2019Acido Retinoico (adh7, cyp26a1, cyp26b1, cyp26c1) o la distribuzione delle loro proteine nell\u2019embrione. Eth da solo non ha indotto effetti avversi sullo sviluppo embrionale. I gruppi esposti a FLUCO 125-250-500\ub5M mostravano specifiche anomalie a livello dell\u2019apparato branchiale, comparabili a quelle precedentemente documentate e correlate ad un probabile aumento di RA. Gli effetti erano dose-dipendenti sia per la frequenza degli embrioni malformati che per la gravit\ue0 delle malformazioni osservate. FLUCO 62.5 \ue8 stata individuata come la NOAEL (No Observed Adverse Effect Level). Gruppi co-esposti a FLUCO ed Eth mostravano un aumento significativo dell\u2019effetto teratogeno rispetto ai gruppi esposti al solo FLUCO. In seguito alla co-esposizione con Eth, FLUCO 62.5 \ub5M mostrava pi\uf9 del 35% degli embrioni anomali. Tale concentrazione non era pi\uf9 identificabile come NOAEL. L\u2019analisi dell\u2019espressione genica ha rivelato che la sola esposizione a Eth o a FLUCO 62.5 \ub5M alterava l\u2019espressione di geni coinvolti nel catabolismo di RA (cyp26a1, cyp26c1) e che tale alterazione peggiorava in seguito alla co-esposizione. La immunocolorazione per CYP26a1 e CYP26c1 non ha evidenziato differenze nelle aree embrionali coinvolte nella loro espressione, che risultavano sovrapponibili nell\u2019area cefalica ai territori di espressione del marker delle cellule delle creste neurali (CRABP1). I dati ottenuti mostrano che la co-esposizione con Eth alla concentrazione sub-teratogena pu\uf2 aumentare il potenziale teratogeno del FLUCO. Questi risultati sono di particolare rilevanza considerando che FLUCO ed Eth non condividono lo stesso meccanismo d\u2019azione: FLUCO interferisce con il catabolismo di RA, mentre Eth potrebbe interferire con la sintesi endogena di RA. L\u2019analisi dell\u2019espressione dei geni correlati al metabolismo di RA suggerisce che l\u2019effetto miscela indotto da FLUCO ed Eth potrebbe essere spiegato da un accumulo di RA dovuto all\u2019azione delle due sostanze. Nella seconda parte di questo studio, embrioni dell\u2019ascidia Ciona intestinalis (Chordata, Tunicata) sono stati esposti in vitro dallo stadio di neurula allo stadio di larva natante a concentrazioni crescenti di FLUCO (31.5-62.5-125-250-500\ub5M), alla concentrazione non effetto di Eth (1\ub5L/mL) o sono state co-esposte a FLUCO 62.5-125-250-500\ub5M ed Eth (1\ub5L/mL). Al termine del periodo di coltura (15 ore) le larve sono state esaminate morfologicamente. Eth da solo non ha indotto effetti avversi sullo sviluppo embrionale, mentre i gruppi esposti a FLUCO mostravano specifiche anomalie a livello delle strutture anteriori, comparabili a quelle gi\ue0 documentate e correlate all\u2019esposizione a RA. \uc8 stato osservato un significativo aumento di larve con malformazioni gravi nei gruppi co-esposti alle miscele di FLUCO ed Eth. I dati preliminari ottenuti sembrano indicare l\u2019ascidia come un adeguato modello alternativo per lo screening teratogeno delle miscele ottenute da fungicidi azolici: viene confermata l\u2019ipotesi di un ruolo chiave dell\u2019Eth nell\u2019aumentare il potenziale teratogeno del FLUCO, puntando l\u2019attenzione su un possibile impatto ambientale dei fungicidi azolici

Effects of Bisphenol A on early development of two ascidian species

Bisphenol A(BPA) is an organic compound present in plastic products that is released into the environment after degradation. BPA is both a teratogenic substance and an endocrine disruptor. The phylogenetic position of tunicates as sister group of vertebrates and their cosmopolitan distribution in marine ecosystems make them reliable model organisms for ecotoxicology bioassays. We tested the effects of different concentration of BPA (0.1, 0.5, 1, 5, 10, and 20 \u3bcM) on sperm viability, fertilization and embryogenesis of two ascidian species, Phallusia mammillata and Ciona intestinalis. We evaluated the type and the incidence of induced malformations. Then we focused on the effects on the nervous system performing animmunostaining of central nervous system (CNS) and whole mount in situ hybridization (WISH) with neural specific markers. Exposure of sperm to BPA did not influence fertilization rate. Co-exposure of eggs and sperm to concentrations higher than 5\u3bcM caused incomplete division of zygotethat did not develop further. In P. mammillata, embryonic development was altered by 20\u3bcM BPA causing a severe phenotype with malformed sensory organs in almost all treated larvae. In C. intestinalis 20 \u3bcM BPA was lethal, while 10 \u3bcM concentration caused alteration to the sensory organs, indicating that C. intestinalis is less tolerant to BPA. Nervous system is a target of BPA action that caused an altered pattern of neural fibers. WISH with Ci-GAD and Ci-TH showed an alterationof dopaminergic and GABAergic cells after exposureto 10 \u3bcM BPA. These results showed that the most sensitive process to BPA is the first cell division. After 2-cell stage, higher concentrations are required to alter the development. BPA confirmed its teratogenic effects on ascidians and its interference with CNS development

Effect of hypoxia-inducible factor-1α on transcription of survivin in non-small cell lung cancer

<p>Abstract</p> <p>Background</p> <p>Survivin is a structurally and functionally unique member of the inhibitor of apoptosis protein (IAP) family. It plays an important role, not only in regulating mitosis but also in inhibiting apoptosis. The current literature contains few reports on the transcriptional regulation of survivin expression in lung cancer.</p> <p>Methods</p> <p>In this study, we investigated the effect of hypoxia-inducible factor-1α (HIF-1α) on the transcriptional activity of the survivin promoter in non-small cell lung cancer (NSCLC). Immunohistochemical staining was used to detect the expression of survivin and HIF-1α in the lung tissue of 120 patients with non-small cell lung cancer (NSCLC) and 40 patients with benign pulmonary disease. We also performed experiments with the lung adenocarcinoma cell line A549 cells, which were cultured under hypoxic conditions. The expression of survivin and HIF-1α was detected by real-time RT-PCR and Western blotting. In the survivin promoter the putative binding-site for HIF-1α, is -19 bp~-16 bp upstream of TSS. We performed site-directed mutagenesis of this binding site, and used luciferase reporter plasmids to determine the relative activity of the survivin promoter in A549 cells. We also studied the effect of HIF-1α on the expression of survivin by dsRNA targeting of HIF-1α mRNA.</p> <p>Results</p> <p>HIF-1α (58.33%) and survivin (81.60%) were both over-expressed in NSCLC and their expressions correlated with one another. They were also expressed in A549 cells under normal and hypoxic conditions, with a significant increase under hypoxic conditions. Site directed mutagenesis of the putative binding site for HIF-1α in the survivin promoter significantly decreased the activity of the survivin promoter in A549 cells. Inhibition of HIF-1α by RNAi decreased the expression of survivin in A549 cell lines.</p> <p>Conclusion</p> <p>Our results indicate that the binding of HIF-1α to the survivin promoter increases transcription of the survivin gene. Thus, HIF-1α is an important transcriptional regulator of survivin expression</p