Long and Very-Long-Chain Ceramides Correlate with A More Aggressive Behavior in Skull Base Chordoma Patients.

BACKGROUND: Skull base chordomas are rare tumors arising from notochord. Sphingolipids analysis is a promising approach in molecular oncology, and it has never been applied in chordomas. Our aim is to investigate chordoma behavior and the role of ceramides. METHODS: Ceramides were extracted and evaluated by liquid chromatography and mass spectrometry in a cohort of patients with a skull base chordoma. Clinical data were also collected and correlated with ceramide levels. Linear regression and correlation analyses were conducted. RESULTS: Analyzing the association between ceramides level and MIB-1, total ceramides and dihydroceramides showed a strong association (r = 0.7257 and r = 0.6733, respectively) with MIB-1 staining (p = 0.0033 and p = 0.0083, respectively). Among the single ceramide species, Cer C24:1 (r = 0.8814, p ≤ 0.0001), DHCer C24:1 (r = 0.8429, p = 0.0002) and DHCer C18:0 (r = 0.9426, p ≤ 0.0001) showed a significant correlation with MIB-1. CONCLUSION: Our lipid analysis showed ceramides to be promising tumoral biomarkers in skull base chordomas. Long- and very-long-chain ceramides, such as Cer C24:1 and DHCer C24:1, may be related to a prolonged tumor survival and aggressiveness, and the understanding of their effective biological role will hopefully shed light on the mechanisms of chordoma radio-resistance, tendency to recur, and use of agents targeting ceramide metabolism.

Determination of the serine palmitoyl transferase inhibitor myriocin by electrospray and Q-trap mass spectrometry.

Myriocin is a potent inhibitor of serine-palmitoyl-transferase, the first and rate-determining enzyme in the sphingolipids biosynthetic pathway. This study developed, validated and applied a LC-MS/MS method to measure myriocin in minute specimens of animal tissue. The chemical analog 14-OH-myriocin was used as the internal standard. The two molecules were extracted from the tissue homogenate by solid-phase extraction, separated by gradient reversed-phase liquid chromatography and measured by negative ion electrospray mass spectrometry in the triple quadrupole. Detection was accomplished by multiple reaction monitoring, employing the most representative transitions, 400@104 and 402@104 for myriocin and 14-OH-myriocin, respectively. The typical limit of detection and lower limit of quantitation of the optimized method were 0.9 pmol/mL (~0.016 pmol injected) and 2.3 pmol/mL, respectively, and the method was linear up to 250 pmol/mL range (r2 = 0.9996). The intra- and between-day repeatability afforded a coefficient of variation ≤7.0%. Applications included quantification of myriocin in mouse lungs after 24 h from administration of ~4 nmol by intra-tracheal delivery. Measured levels ranged from 4.11 (median; 2.3-7.4 IQR, n = 4) to 11.7 (median; 7.6-22.7 interquartile range (IQR), n = 6) pmol/lung depending on the different formulations used. Myriocin was also measured in retinas of mice treated by intravitreal injection and ranged from 0.045 (less than the limit of detection) to 0.35 pmol/retina.

Application of An Improved HPLC-FL Method to Screen Serine Palmitoyl Transferase Inhibitors.

In this work, we reported the application and validation of an improved high-performance liquid chromatography method coupled with a fluorimetric detector (HPLC-FL) to screen the activity of two heterocyclic derivatives reported as serine palmitoyl transferase (SPT) inhibitors. The analytical conditions were optimized in terms of the derivatization procedure, chromatographic condition, extraction procedure, and method validation according to EMEA guidelines. Once fully optimized, the method was applied to assess the SPT-inhibitory activity of the above-mentioned derivatives and of the reference inhibitor myriocin. The obtained results, expressed as a percentage of residual SPT activity, were compared to those obtained with the reference radio immune assay (RIA). The good correlation between the two types of assay demonstrated that the improved HPLC-FL method is suitable for a preliminary and rapid screening of potential SPT-inhibitors.

Patient report outcomes in cryoballoon ablation of atrial fibrillation during the COVID Era: Insights from the 1STOP project.

BACKGROUND: Pulmonary vein isolation by cryoablation (PVI-C) is a standard therapy for the treatment of patients with symptomatic atrial fibrillation (AF). AF symptoms are highly subjective; however, they are important outcomes for the patient. The aim is to describe the use and impact of a web-based App to collect AF-related symptoms in a population of patients who underwent PVI-C in seven Italian centers. METHODS: A patient App to collect AF-related symptoms and general health status was proposed to all patients who underwent an index PVI-C. Patients were divided into two groups according to the utilization of the App or the non-usage. RESULTS: Out of 865 patients, 353 (41%) subjects composed the App group, and 512 (59%) composed the No-App group. Baseline characteristics were comparable between the two cohorts except for age, sex, type of AF, and body mass index. During a mean follow-up of 7.9±13.8 months, AF recurrence was found in 57/865 (7%) subjects with an annual rate of 7.36% (95% CI:5.67-9.55%) in the No-App versus 10.99% (95% CI:9.67-12.48%) in the App group, p=0.007. In total, 14,458 diaries were sent by the 353 subjects in the App group and 77.1% reported a good health status and no symptoms. In only 518 diaries (3.6%), the patients reported a bad health status, and bad health status was an independent parameter of AF recurrence during follow-up. CONCLUSIONS: The use of a web App to record AF-related symptoms was feasible and effective. Additionally, a bad health status reporting in the App was associated with AF recurrence during follow-up.

Performance of transcatheter pacing system use in relation to patients' age.

PURPOSE: Real-world safety data on the use of transcatheter pacing systems particularly in very elderly patients is still limited. The aim of this analysis was to investigate the effect of age on the safety and efficacy of leadless pacemaker implant. METHODS: From May 2016 through July 2019, 577 patients were implanted with a leadless single-chamber pacemaker according to current pacing indication in 15 Italian cardiologic centers. The population was divided into age quartiles for evaluation, including (1) < 70 years, (2) 70-77 years, (3) 78-83 years, and (4) ≥ 83 years. Procedural data, complications, and electrical parameters were collected at baseline and during the follow-up. RESULTS: Procedural-related complication occurrence was very low (< 1.0%) and similar in the four subgroups according to age even if the older patients were more frail. No cardiac tamponade was reported. Among the groups, no difference was observed in procedural time, fluoroscopy time duration, and electrical parameters (mean pacing impedance: 750 ± 192 and 599 ± 156, mean pacing threshold: 0.7 ± 0.5 and 0.7 ± 0.6, and mean right ventricular sensing 10.7 ± 6.1 and 11.5 ± 4.8 at implant and last follow-up, respectively). CONCLUSIONS: The reported data demonstrated a high degree of safety during leadless implant across all patient ages. Procedural complications and device electrical measurements were similar among the different ages.

Efficacy of a tool combining guide-wire and stylet for the left ventricular lead positioning.

AIMS: The left ventricular (LV) lead for cardiac resynchronization therapy (CRT) is usually positioned in the coronary sinus via a stylet-guided or an 'over-the-wire' approach. Recently, a new tool has been developed, the Medtronic Attain Hybrid, that combines guide-wire and stylet features. We assessed its safety and efficacy in comparison with standard tools currently used in clinical practice. METHODS AND RESULTS: Patients undergoing standard CRT device implantation were enrolled in seven Italian centres. In the preliminary phase of the study (Phase I), data were collected during implantation procedures performed with standard tools (three patients per centre). Subsequently, the Attain Hybrid was made available in the centres and data were collected for all consecutive patients undergoing implantation during the following year. A learning phase was considered (Phase II), and the last three patients per centre (Phase III) were used for comparison with Phase I. One hundred and seventeen patients were enrolled: 21 patients in Phase I, 75 in Phase II, and 21 in Phase III. Rates of successful implantation were similar in Phases I and III (95 vs. 100%, P=1.000). The pre-defined target vein was reached in 15 (71%) patients in Phase I and in 21 (100%) patients in Phase III (P=0.021). In 10 (48%) procedures during Phase I, LV lead positioning necessitated switching from guide-wire to stylet; this proportion decreased during Phase III (14%, P=0.043). Mean LV positioning time was 16±7 min in Phase I and 11±6 min in Phase III (P=0.040). No adverse events or lead-related complications were detected on implantation or during a follow-up of 6±4 months. CONCLUSION: The Attain Hybrid is safe and effective. It significantly improves target vein accessibility and reduces procedural time in comparison with conventional tools.

Effect of organic co-solvents in the evaluation of the hydroxyl radical scavenging activity by the 2-deoxyribose degradation assay: The paradigmatic case of α-lipoic acid.

The 2-deoxyribose degradation assay (2-DR assay) is an in vitro method broadly used for evaluating the scavenging activity against the hydroxyl radical (HO). One of the major drawbacks of the assay, however, is that only water soluble compounds can be tested for their radical-scavenging activity. Lipoic acid (LA) is an excellent scavenger of HO but it exhibits a low solubility in the aqueous milieu of the 2-DR assay and a high tendency to polymerize under a variety of conditions. We used LA as a paradigmatic substrate to evaluate the effect of several organic co-solvents in increasing its solubility. Most of these solvents, however, demonstrated to be potent scavengers of HO making their use in the 2-DR assay improper. On the other hand, acetonitrile showed a remarkably low reactivity toward HO (rate constant ~8.7×106M-1s-1) which allowed us to use it as a co-solvent in the preparation of stock solutions of LA ~5mM. We therefore evaluated the radical-scavenging activity of LA by the 2-DR assay in a relatively large range of concentrations, 1-200µM. We found that the rate constant for LA+HO is diffusion-controlled (~1×1010M-1s-1 in water at 25°C) and uninfluenced by the presence of small quantities of acetonitrile. Therefore, the use of acetonitrile in the 2-DR assay does not interfere with the test and may increase the solubility of the radical scavengers.

Inhibitors of ceramide de novo biosynthesis rescue damages induced by cigarette smoke in airways epithelia.

Exposure to cigarette smoke represents the most important risk factor for the development of chronic obstructive pulmonary disease (COPD). COPD is characterized by chronic inflammation of the airways, imbalance of proteolytic activity resulting in the destruction of lung parenchyma, alveolar hypoxia, oxidative stress, and apoptosis. Sphingolipids are structural membrane components whose metabolism is altered during stress. Known as apoptosis and inflammation inducer, the sphingolipid ceramide was found to accumulate in COPD airways and its plasma concentration increased as well. The present study investigates the role of sphingolipids in the cigarette smoke-induced damage of human airway epithelial cells. Lung epithelial cells were pre-treated with sphingolipid synthesis inhibitors (myriocin or XM462) and then exposed to a mixture of nicotine, acrolein, formaldehyde, and acetaldehyde, the major toxic cigarette smoke components. The inflammatory and proteolytic responses were investigated by analysis of the mRNA expression (RT-PCR) of cytokines IL-1ß and IL-8, and matrix metalloproteinase-9 and of the protein expression (ELISA) of IL-8. Ceramide intracellular amounts were measured by LC-MS technique. Ferric-reducing antioxidant power test and superoxide anion radical scavenging activity assay were used to assess the antioxidant power of the inhibitors of ceramide synthesis. We here show that ceramide synthesis is enhanced under treatment with a cigarette smoke mixture correlating with increased expression of inflammatory cytokines and matrix metalloproteinase 9. The use of inhibitors of ceramide synthesis protected from smoke induced damages such as inflammation, oxidative stress, and proteolytic imbalance in airways epithelia.