Lipidomics reveals the reshaping of the mitochondrial phospholipid profile in cells lacking OPA1 and mitofusins.

Depletion or mutations of key proteins for mitochondrial fusion, like optic atrophy 1 (OPA1) and mitofusins 1 and 2 (Mfn 1 and 2), are known to significantly impact the mitochondrial ultrastructure, suggesting alterations of their membranes' lipid profiles. In order to make an insight into this issue, we used hydrophilic interaction liquid chromatography coupled with electrospray ionization-high resolution MS to investigate the mitochondrial phospholipid (PL) profile of mouse embryonic fibroblasts knocked out for OPA1 and Mfn1/2 genes. One hundred sixty-seven different sum compositions were recognized for the four major PL classes of mitochondria, namely phosphatidylcholines (PCs, 63), phosphatidylethanolamines (55), phosphatidylinositols (21), and cardiolipins (28). A slight decrease in the cardiolipin/PC ratio was found for Mfn1/2-knockout mitochondria. Principal component analysis and hierarchical cluster analysis were subsequently used to further process hydrophilic interaction liquid chromatography-ESI-MS data. A progressive decrease in the incidence of alk(en)yl/acyl species in PC and phosphatidylethanolamine classes and a general increase in the incidence of unsaturated acyl chains across all the investigated PL classes was inferred in OPA1 and Mfn1/2 knockouts compared to WT mouse embryonic fibroblasts. These findings suggest a reshaping of the PL profile consistent with the changes observed in the mitochondrial ultrastructure when fusion proteins are absent. Based on the existing knowledge on the metabolism of mitochondrial phospholipids, we propose that fusion proteins, especially Mfns, might influence the PL transfer between the mitochondria and the endoplasmic reticulum, likely in the context of mitochondria-associated membranes.

HILIC-ESI-MS analysis of phosphatidic acid methyl esters artificially generated during lipid extraction from microgreen crops.

The uncontrolled activation of endogenous enzymes may introduce both qualitative and quantitative artefacts when lipids are extracted from vegetal matrices. In the present study, a method based on hydrophilic interaction liquid chromatography coupled either to high-resolution/accuracy Fourier-transform mass spectrometry (HILIC-ESI-FTMS) or to linear ion trap multiple stage mass spectrometry (HILIC-ESI-MSn , with n = 2 and 3) with electrospray ionization was developed to unveil one of those artefacts. Specifically, the artificial generation of methyl esters of phosphatidic acids (MPA), catalysed by endogenous phospholipase D (PLD) during lipid extraction from five oleaginous microgreen crops (chia, soy, flax, sunflower and rapeseed), was studied. Phosphatidylcholines (PC) and phosphatidylglycerols (PG) were found to be the most relevant precursors of MPA among glycerophospholipids (GPLs), being involved in a transphosphatidylation process catalysed by PLD and having methanol as a coreactant. The combination of MS2 and MS3 measurements enabled the unambiguous recognition of MPA from their fragmentation pathways, leading to distinguish them from isobaric PA including a further CH2 group on their side chains. PLD was also found to catalyse the hydrolysis of PC and PG to phosphatidic acids (PAs). The described transformations were confirmed by the remarkable decrease of MPA abundance observed when isopropanol, known to inhibit PLD, was tentatively adopted instead of water during the homogenization of microgreens. The unequivocal identification of MPA might be exploited to assess if GPL alterations are actually triggered by endogenous PLD during lipid extractions from specific vegetal tissues.

Pain management policies and practices in pediatric emergency care: a nationwide survey of Italian hospitals.

BACKGROUND: Pain experienced by children in emergency departments (EDs) is often poorly assessed and treated. Although local protocols and strategies are important to ensure appropriate staff behaviours, few studies have focussed on pain management policies at hospital or department level. This study aimed at describing the policies and reported practices of pain assessment and treatment in a national sample of Italian pediatric EDs, and identifying the assocoated structural and organisational factors. METHODS: A structured questionnaire was mailed to all the 14 Italian pediatric and maternal and child hospitals and to 5 general hospitals with separate pediatric emergency room. There were no refusals. Information collected included the frequency and mode of pain assessment, presence of written pain management protocols, use of local anaesthetic (EMLA cream) before venipuncture, and role of parents. General data on the hospital and ED were also recorded. Multiple Correspondence Analysis was used to explore the multivariable associations between the characteristics of hospitals and EDs and their pain management policies and practices. RESULTS: Routine pain assessment both at triage and in the emergency room was carried out only by 26% of surveyed EDs. About one third did not use algometric scales, and almost half (47.4%) did not have local protocols for pain treatment. Only 3 routinely reassessed pain after treatment, and only 2 used EMLA. All EDs allowed parents' presence and most (17, 89.9%) allowed them to stay when painful procedures were carried out. Eleven hospitals (57.9%) allowed parents to hold their child during blood sampling. Pediatric and maternal and child hospitals, those located in the North of Italy, equipped with medico-surgical-traumatological ED and short stay observation, and providing full assessment triage over 24 hours were more likely to report appropriate policies for pain management both at triage and in ER. A nurses to admissions ratio ≥ median was associated with better pain management at triage. CONCLUSIONS: Despite availability of national and international guidelines, pediatric pain management is still sub-optimal in Italian emergency departments. Multifaceted strategies including development of local policies, staff educational programs, and parental involvement in pain assessment should be carried out and periodically reinforced.

Parental involvement and kangaroo care in European neonatal intensive care units: a policy survey in eight countries.

OBJECTIVE: To compare, in a large representative sample of European neonatal intensive care units, the policies and practices regarding parental involvement and holding babies in the kangaroo care position as well as differences in the tasks mothers and fathers are allowed to carry out. DESIGN: Prospective multicenter survey. SETTING: Neonatal intensive care units in eight European countries (Belgium, Denmark, France, Italy, The Netherlands, Spain, Sweden, and the United Kingdom). PATIENTS: Patients were not involved in this study. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A structured questionnaire was mailed to 362 units (response rate 78%); only units with ≥50 very-low-birth-weight annual admissions were considered for this study. Facilities for parents such as reclining chairs near the babies' cots, beds, and a dedicated room were common, but less so in Italy and Spain. All units in Sweden, Denmark, the United Kingdom, and Belgium reported encouraging parental participation in the care of the babies, whereas policies were more restrictive in Italy (80% of units), France (73%), and Spain (41%). Holding babies in the kangaroo care position was widespread. However, in the United Kingdom, France, Italy, and Spain, many units applied restrictions regarding its frequency (sometimes or on parents request only, rather than routinely), method (conventional rather than skin-to-skin), and clinical conditions (especially mechanical ventilation and presence of umbilical lines) that would prevent its practice. In these countries, fathers were routinely offered kangaroo care less frequently than mothers (p < .001) and less often it was skin-to-skin (p < .0001). CONCLUSIONS: This study showed that, although the majority of units in all countries reported a policy of encouraging both parents to take part in the care of their babies, the intensity and ways of involvement as well as the role played by mothers and fathers varied within and between countries.

Heel blood sampling in European neonatal intensive care units: compliance with pain management guidelines.

OBJECTIVE: To describe the use of heel blood sampling and non-pharmacological analgesia in a large representative sample of neonatal intensive care units (NICUs) in eight European countries, and compare their self-reported practices with evidence-based recommendations. METHODS: Information on use of heel blood sampling and associated procedures (oral sweet solutions, non-nutritive sucking, swaddling or positioning, topical anaesthetics and heel warming) were collected through a structured mail questionnaire. 284 NICUs (78% response rate) participated, but only 175 with ≥50 very low birth weight admissions per year were included in this analysis. RESULTS: Use of heel blood sampling appeared widespread. Most units in the Netherlands, UK, Denmark, Sweden and France predominantly adopted mechanical devices, while manual lance was still in use in the other countries. The two Scandinavian countries and France were the most likely, and Belgium and Spain the least likely to employ recommended combinations of evidence-based pain management measures. CONCLUSIONS: Heel puncture is a common procedure in preterm neonates, but pain appears inadequately treated in many units and countries. Better compliance with published guidelines is needed for clinical and ethical reasons.