[Out-of-hospital cardiac arrest: University of Trieste students' skills on resuscitative maneuvers and automated external defibrillator]. / Arresto cardiaco extraospedaliero: le competenze degli studenti dell'Università degli Studi di Trieste sulle manovre rianimatorie e l'utilizzo del defibrillatore automatico esterno.

BACKGROUND: Out-of-hospital cardiac arrest is the third leading cause of death in industrialized countries. Although most cardiac arrests are witnessed, survival is only 2-10%, since bystanders are often unable to correctly perform cardiopulmonary resuscitation (CPR). This study aims to assess the theoretical and practical knowledge of CPR and the use of the automatic external defibrillator in university students. METHODS: The study involved a total of 1686 students from 21 different faculties of the University of Trieste, 662 students from healthcare faculties and 1024 from non-healthcare faculties. Basic life support and early defibrillation (BLS-D) courses and retrainings after 2 years are mandatory for students in their final 2-year healthcare faculties at the University of Trieste. Through the platform "EUSurvey" from March to June 2021, they were given an online questionnaire of 25 multiple choice questions to investigate the performance of BLS-D. RESULTS: In the overall population, 68.7% knew how to diagnose a cardiac arrest and 47.5% knew the time frame after which irreversible brain damage occurs. Practical knowledge was analyzed by evaluating the correct answers to all four questions on performing CPR (i.e. hand position during compressions, frequency of compressions, depth of compressions, and ventilation-compression ratio). Health faculties students have better theoretical and practical knowledge of CPR than their colleagues of non-healthcare faculties, with better overall knowledge on the all four practical questions (11.2% vs 4.3%; p<0.001). Final-year medical students of the University of Trieste, who attended the BLS-D course and underwent retraining after 2 years, have achieved better results than first year medical students (without BLS-D training) (38.1% vs 2.7%; p<0.001). CONCLUSION: Mandatory BLS-D training and retraining leads to a better knowledge of cardiac arrest management and consequently a better patient outcome. In order to improve patient survival, heartsaver (BLS-D for laics) training should be extended as obligatory in all university courses.

Cog2 null mutant CHO cells show defective sphingomyelin synthesis.

The COG (conserved oligomeric Golgi complex) is a Golgi-associated tethering complex involved in retrograde trafficking of multiple Golgi enzymes. COG deficiencies lead to misorganization of the Golgi, defective trafficking of glycosylation enzymes, and abnormal N-, O- and ceramide-linked oligosaccharides. Here, we show that in Cog2 null mutant ldlC cells, the content of sphingomyelin (SM) is reduced to ∼25% of WT cells. Sphingomyelin synthase (SMS) activity is essentially normal in ldlC cells, but in contrast with the typical Golgi localization in WT cells, in ldlC cells, transfected SMS1 localizes to vesicular structures scattered throughout the cytoplasm, which show almost no signal of co-transfected ceramide transfer protein (CERT). Cog2 transfection restores SM formation and the typical SMS1 Golgi localization phenotype. Adding exogenous N-6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl-4-d-erythro-sphingosine (C(6)-NBD-ceramide) to ldlC cell cultures results in normal SM formation. Endogenous ceramide levels were 3-fold higher in ldlC cells than in WT cells, indicating that Golgi misorganization caused by Cog2 deficiency affects the delivery of ceramide to sites of SM synthesis by SMS1. Considering the importance of SM as a structural component of membranes, this finding is also worth of consideration in relation to a possible contribution to the clinical phenotype of patients suffering congenital disorders of glycosylation type II.

Defective GM3 synthesis in Cog2 null mutant CHO cells associates to mislocalization of lactosylceramide sialyltransferase in the Golgi complex.

The conserved oligomeric Golgi (COG) complex is a eight subunit (COG1 to 8) tethering complex involved in the retrograde trafficking of multiple Golgi processing proteins. Here we studied the glycolipid synthesis status in ldlC cells, a Cog2 null mutant CHO cell line. Biochemical studies revealed a block in the coupling between LacCer and GM3 synthesis, resulting in decreased levels of GM3 in these cells. Uncoupling was not attributable to decreased activity of the glycosyltransferase that uses LacCer as acceptor substrate (SialT1). Rather, immunocytochemical experiments evidenced a mislocalization of SialT1 as consequence of the lack of Cog2 in these cells. Co-immunoprecipitation experiments disclose a Cog2 mediated interaction of SialT1 with the COG complex member Cog1. Results indicate that cycling of some Golgi glycolipid glycosyltransferases depends on the participation of the COG complex and that deficiencies in COG complex subunits, by altering their traffic and localization, affect glycolipid composition.

Modulation of GalT1 and SialT1 sub-Golgi localization by SialT2 expression reveals an organellar level of glycolipid synthesis control.

Ganglioside glycosyltransferases organize as multienzyme complexes that localize in different sub-Golgi compartments. Here we studied whether in CHO-K1 cells lacking CMP-NeuAc: GM3 sialyltransferase (SialT2), the sub-Golgi localization of UDP-Gal:glucosylceramide beta-1,4-galactosyltransferase (GalT1) and CMP-NeuAc:lactosylceramide sialyltransferase (SialT1) complex is affected when SialT2, another member of this complex, is coexpressed. GalT1 and SialT1 sub-Golgi localization was determined by studying the effect of brefeldin A (BFA) and monensin on the synthesis of glycolipids and on the sub-Golgi localization of GalT1(1-52)-CFP (cyan fluorescent protein) and SialT1(1-54)-YFP (yellow fluorescent protein) chimeras by single cell fluorescence microscopy and by isopycnic subfractionation. We found that BFA, and also monensin, impair the synthesis of glycolipids beyond GM3 ganglioside in wild type (WT) cells but beyond GlcCer in SialT2(+) cells. Although BFA redistributed GalT1-CFP and SialT1-YFP to the endoplasmic reticulum in WT cells, a fraction of these chimeras remained associated with a distal Golgi compartment, enriched in trans Golgi network, and recycling endosome markers in SialT2(+) cells. In BFA-treated cells, the percentage of GalT1-CFP and SialT1-YFP associated with Golgi-like membrane fractions separated by isopycnic subfractionation was higher in SialT2(+) cells than in WT cells. These effects were reverted by knocking down the expression of SialT2 with specific siRNA. Results indicate that sub-Golgi localization of glycosyltransferase complexes may change according to the relative levels of the expression of participating enzymes and reveal a capacity of the organelle to adapt the topology of the glycolipid synthesis machinery to functional states of the cell.

Cytoplasmic tails of SialT2 and GalNAcT impose their respective proximal and distal Golgi localization.

Complex glycolipid synthesis is catalyzed by different glycosyltransferases resident of the Golgi complex. Most of them are type II membrane proteins comprising a lumenal, C-terminal domain linked to an N-terminal domain (Ntd) constituted by a short cytoplasmic tail (ct), a transmembrane, and a lumenal stem regions. They concentrate selectively in different sub-Golgi compartments, in an overlapped manner, acting in succession in the addition of sugars to acceptor glycolipids. The Ntds are sufficient to localize glycosyltransferases in the Golgi complex, but it is not clear whether they also confer selective concentration in sub-Golgi compartments. Here, we studied whether the Ntd of SialT2, localized in the proximal Golgi, and the one of GalNAcT, a trans/TGN Golgi-concentrated enzyme, concentrate reporter proteins in the corresponding sub-Golgi compartment. The sub-Golgi concentration of the Ntds fused to spectral variants of the GFP was determined in CHO-K1 cells from their behavior upon addition of brefeldin A. Fluorescence microscopy and subcellular fractionation showed that the SialT2 Ntd concentrates in a proximal sub-Golgi compartment - and that of GalNAcT in TGN elements. Exchanging the transmembrane region and the cts of SialT2 and GalNAcT indicates that information for proximal or distal Golgi concentration is associated with the cts.