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One of the most widely used strategies for metabolite annotation in untargeted LCMS is based on the analysis of MSn spectra acquired using data-dependent acquisition (DDA), where precursor ions are sequentially selected from MS scans based on user-selected criteria. However, the number of MSn spectra that can be acquired during a chromatogram is limited and a trade-off between analytical speed, sensitivity and coverage must be ensured. In this research, we compare four different strategies for automated MS2 DDA, which can be easily implemented in the frame of standard QA/QC workflows for untargeted LC–MS. These strategies consist of (i) DDA in the MS working range; (ii) iterated DDA split into several m/z intervals; (iii) dynamic iterated DDA of (pre)selected potentially informative features; and (iv) dynamic iterated DDA of (pre)annotated metabolic features using a reference database. Their performance was assessed using the analysis of human milk samples as model example by comparing the percentage of LC–MS features selected as the precursor ion for MS2, the number, and class of annotated features, the speed and confidence of feature annotation, and the number of LC runs required.

Pregnancy induces a number of immunological, hormonal, and metabolic changes that are necessary for the mother to adapt her body to this new physiological situation. The microbiome of the mother, the placenta and the fetus influence the fetus growth and undoubtedly plays a major role in the adequate development of the newborn infant. Hence, the microbiome modulates the inflammatory mechanisms related to physiological and pathological processes that are involved in the perinatal progress through different mechanisms. The present review summarizes the actual knowledge related to physiological changes in the microbiota occurring in the mother, the fetus, and the child, both during neonatal period and beyond. In addition, we approach some specific pathological situations during the perinatal periods, as well as the influence of the type of delivery and feeding.

Background Breast milk is a vehicle to transfer protective antibodies from the lactating mother to the neonate. After SARS-CoV-2 infection, virus-specific IgA and IgG have been identified in breast milk, however, there are limited data on the impact of different COVID-19 vaccine types in lactating women. This study is aimed to evaluate the time course of induction of SARS-CoV-2-specific IgA and IgG in breast milk after vaccination. Methods In this prospective observational study in Spain, 86 lactating women from priority groups receiving the vaccination against SARS-CoV-2 were included. Breast milk samples were collected longitudinally at seven or eight-time points (depending on vaccine type). A group with confirmed SARS-CoV-2 infection ( n =19) and a group of women from pre-pandemic time ( n =20) were included for comparison. Results Eighty-six vaccinated lactating women [mean age, 34.6 ± 3.7 years] of whom 96% were Caucasian and 92% were healthcare workers. A total number of 582 milk samples were included, and vaccine distribution was BioNTech/Pfizer (BNT162b2, n =34), Moderna (mRNA-1273, n =20), and AstraZeneca (ChAdOx1 nCoV-19, n =32). For each vaccine, 7 and 8 longitudinal time points were collected from baseline up to 30 days after the second dose for mRNA vaccines and adenovirus-vectored vaccines, respectively. A strong reactivity was observed for IgG and IgA after vaccination mainly after the 2 nd dose. The presence and persistence of specific SARS-CoV-2 antibodies in breast milk were dependent on the vaccine type, with higher IgG and IgA levels in mRNA-based vaccines when compared to AstraZeneca, and on previous virus exposure. High intra- and inter-variability were observed, being relevant for IgA antibodies. In milk from vaccinated women, anti-SARS-CoV-2 IgG was significantly higher while IgA levels were lower than in milk from COVID-19-infected women. Women with previous COVID-19 increased their IgG antibodies levels after the first dose to a similar level observed in vaccinated women after the second dose. Conclusions COVID-19 vaccination induced anti-SARS-CoV-2 IgA and IgG in breast milk with higher levels after the 2 nd dose. Levels of anti-SARS-CoV-2 IgA and IgG are dependent on the vaccine type. Further studies are warranted to demonstrate the protective antibody effect against COVID-19 in infants from vaccinated and infected mothers. Trial registration NCT04751734 (date of registration is on February 12, 2021)

ObjectivesTo develop and validate a specific protocol for SARS-CoV-2 detection in breast milk matrix and to determine the impact of maternal SARS-CoV-2 infection on the presence, concentration and persistence of specific SARS-CoV-2 antibodies.Design and patientsThis is a prospective, multicentre longitudinal study (April–December 2020) in 60 mothers with SARS-CoV-2 infection and/or who have recovered from COVID-19. A control group of 13 women before the pandemic were also included.SettingSeven health centres from different provinces in Spain.Main outcome measuresPresence of SARS-CoV-2 RNA in breast milk, targeting the N1 region of the nucleocapsid gene and the envelope (E) gene; presence and levels of SARS-CoV-2-specific immunoglobulins (Igs)—IgA, IgG and IgM—in breast milk samples from patients with COVID-19.ResultsAll breast milk samples showed negative results for presence of SARS-CoV-2 RNA. We observed high intraindividual and interindividual variability in the antibody response to the receptor-binding domain of the SARS-CoV-2 spike protein for each of the three isotypes IgA, IgM and IgG. Main Protease (MPro) domain antibodies were also detected in milk. 82.9% (58 of 70) of milk samples were positive for at least one of the three antibody isotypes, with 52.9% of these positive for all three Igs. Positivity rate for IgA was relatively stable over time (65.2%–87.5%), whereas it raised continuously for IgG (from 47.8% for the first 10 days to 87.5% from day 41 up to day 206 post-PCR confirmation).ConclusionsOur study confirms the safety of breast feeding and highlights the relevance of virus-specific SARS-CoV-2 antibody transfer. This study provides crucial data to support official breastfeeding recommendations based on scientific evidence. Trial registration number NCT04768244.

Aerobic metabolism is highly efficient in providing energy for multicellular organisms. However, even under physiological conditions, an incomplete reduction of oxygen produces reactive oxygen species and, subsequently, oxidative stress. Some of these chemical species are highly reactive free radicals capable of causing functional and structural damage to cell components (protein, lipids, or nucleotides). Oxygen is the most used drug in ill-adapted patients during the newborn period. The use of oxygen may cause oxidative stress-related diseases that increase mortality and cause morbidity with adverse long-term outcomes. Conditions such as prematurity or birth asphyxia are frequently treated with oxygen supplementation. Both pathophysiological situations of hypoxia–reoxygenation in asphyxia and hyperoxia in premature infants cause a burst of reactive oxygen species and oxidative stress. Recently developed analytical assays using mass spectrometry have allowed us to determine highly specific biomarkers with minimal samples. The detection of these metabolites will help improve the diagnosis, evolution, and response to therapy in oxidative stress-related conditions during the newborn period.

This research was funded by LaMarató-TV3 (DIM-2-ELI, ref. 2018-27/30-31) and also by the European Research Council under the European Union’s Horizon 2020 research and innovation program (ERC starting grant, no. 639226). K.R-A holds a fellowship from the Spanish Ministry of Economy, Industry and Competitiveness (FPU 19/05150).

Human milk (HM) is considered the gold standard for infant nutrition. HM contains macro- and micronutrients, as well as a range of bioactive compounds (hormones, growth factors, cell debris, etc.). The analysis of the complex and dynamic composition of HM has been a permanent challenge for researchers. The use of novel, cutting-edge techniques involving different metabolomics platforms has permitted to expand knowledge on the variable composition of HM. This review aims to present the state-of-the-art in untargeted metabolomic studies of HM, with emphasis on sampling, extraction and analysis steps. Workflows available from the literature have been critically revised and compared, including a comprehensive assessment of the achievable metabolome coverage. Based on the scientific evidence available, recommendations for future untargeted HM metabolomics studies are included.

Background and Objectives. Neonatal sepsis is a serious condition with a high rate of mortality and morbidity. Currently, the gold standard for sepsis diagnosis is a positive blood culture, which takes 48–72 h to yield results. We hypothesized that identifying differentially expressed miRNA pattern in neonates with late-onset Gram-positive sepsis would help with an earlier diagnosis and therapy. Methods. This is a prospective observational study in newborn infants with late-onset Gram positive bacterial sepsis and non-septic controls. Complementary to blood culture, an aliquot of 0.5 mL of blood was used to determine small non-coding RNA expression profiling using the GeneChip miRNA 4.0 Array. Results. A total of 11 very low birth-weight neonates with late-onset Gram-positive sepsis and 16 controls were analyzed. Further, 217 differentially expressed miRNAs were obtained between both groups. Subsequently, a combined analysis was performed with these miRNAs and 4297 differentially expressed genes. We identified 33 miRNAs that regulate our mRNAs, and the most relevant biological processes are associated with the immune system and the inflammatory response. Conclusions. The miRNA profiling in very low birth-weight neonates distinguishes late-onset Gram-positive sepsis versus control neonates.

The glutathione (GSH)-to-glutathione disulfide (GSSG) ratio is an essential node contributing to intracellular redox status. GSH/GSSG determination in whole blood can be accomplished by liquid chromatography–mass spectrometry (LC-MS) after the derivatization of GSH with N-ethylmaleimide (NEM). While this is feasible in a laboratory environment, its application in the clinical scenario is cumbersome and therefore ranges reported in similar populations differ noticeably. In this work, an LC-MS procedure for the determination of GSH and GSSG in dried blood spot (DBS) samples based on direct in situ GSH derivatization with NEM of only 10 µL of blood was developed. This novel method was applied to 73 cord blood samples and 88 residual blood volumes from routine newborn screening performed at discharge from healthy term infants. Two clinical scenarios simulating conditions of sampling and storage relevant for routine clinical analysis and clinical trials were assessed. Levels of GSH-NEM and GSSG measured in DBS samples were comparable to those obtained by liquid blood samples. GSH-NEM and GSSG median values for cord blood samples were significantly lower than those for samples at discharge. However, the GSH-NEM-to-GSSG ratios were not statistically different between both groups. With DBS testing, the immediate manipulation of samples by clinical staff is reduced. We therefore expect that this method will pave the way in providing an accurate and more robust determination of the GSH/GSSG values and trends reported in clinical trials.

This is a narrative review about the mechanisms involved in bacterial sepsis in preterm infants, which is an illness with a high incidence, morbidity, and mortality. The role of the innate immune response and its relationship with oxidative stress in the pathogenesis are described as well as their potential implementation as early biomarkers. Moreover, we address the impact that all the mechanisms triggered by sepsis have on the dysbiosis and the changes on neonatal microbiota.