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Background and AimsPrevious trials evaluating remote ischaemic preconditioning in children undergoing cardiac surgery showed mixed results. We sought to determine whether adequately delivered bilateral preconditioning is cardioprotective in young children, with or without cyanosis, undergoing surgery.MethodsProspective, double-blind, randomised controlled trial at two UK centres. Children aged 3-36 months undergoing tetralogy of Fallot repair or ventricular septal defect closure were randomised in a 1:1 ratio to receive either bilateral preconditioning or sham intervention. Participants were followed up until hospital discharge or 30 days. The primary outcome was area under the curve for high-sensitivity troponin-T in the first 24 hours after surgery, analysed by intention-to-treat. Right atrial biopsies were obtained in selected patients. Trial registration:ISRCTN12923441.ResultsBetween 24 October 2016 and 8 December 2020, 120 eligible children were randomised to receive either bilateral preconditioning (n=60) or sham intervention (n=60). Participants had a median age of 7 months and 42 (35%) were female. The primary outcome, area under the curve for hs-troponin-T was higher in the preconditioning group (mean: 70.0±50.9µg/L/hr, n=56) than in controls (mean: 55.6±30.1µg/L/hr, n=58), p=0.04. Sub-group analyses did not show a differential treatment effect by oxygen saturations (pinteraction=0.25) but showed evidence of differential treatment effect by underlying defect (pinteraction=0.04). Myocardial metabolism, quantified in atrial biopsies, and secondary outcomes were not different between randomised groups.ConclusionsBilateral remote ischemic preconditioning does not attenuate myocardial injury in children undergoing surgical repair for congenital heart defects, and there was evidence of potential harm in unstented tetralogy of Fallot.

The tricarboxylic acid (TCA) cycle, or Krebs cycle, is the central pathway of energy production in eukaryotic cells and plays a key part in aerobic respiration throughout all kingdoms of life. The enzymes involved in this cycle generate the reducing equivalents NADH and FADH2 by a series of enzymatic reactions, which are utilized by the electron transport chain to produce ATP. One of the pivotal enzymes in this cycle is 2-oxoglutarate dehydrogenase complex (OGDHC), which generates NADH by oxidative decarboxylation of 2-oxoglutarate to succinyl-CoA. OGDHC is a megadalton protein complex originally thought to be assembled just from three catalytically active subunits (E1o, E2o, E3). In fungi and animals, however, the protein MRPS36 has more recently been proposed as a putative additional component. Based on extensive XL-MS data obtained from measurements in mice and bovine heart mitochondria, supported by phylogenetic analyses, we provide evidence that MRPS36 is an essential member of OGDHC, albeit only in eukaryotes. Comparative sequence analysis and computational structure predictions reveal that in eukaryotic OGDHC, E2o does not contain the peripheral subunit-binding domain (PSBD), present in bacterial and archaeal E2o’s. We propose that in eukaryotes MRPS36 evolved as an E3 adaptor protein, functionally replacing the PSBD. We further provide a refined structural model of the complete eukaryotic OGDHC containing 16 E1o, 12 E3, and 6 subunits of MRPS36 accommodated around the OGDHC core composed of 24 E2o subunits (3.45 MDa). The model provides new insights into the OGDH complex topology and stipulates putative mechanistic implications.

Acute myeloid leukaemia (AML) creates an immunosuppressive environment to conventional T cells through Arginase 2 (ARG2)-induced arginine depletion. We identify that AML blasts release the acute phase protein serum amyloid A (SAA), which acts in an autocrine manner to upregulate ARG2 expression and activity, and promote AML blast viability. Following in vitro cross-talk invariant natural killer T (iNKT) cells become activated, upregulate mitochondrial capacity, and release IFN-γ. iNKT retain their ability to proliferate and be activated despite the low arginine AML environment, due to the upregulation of Large Neutral Amino Acid Transporter-1 (LAT-1) and Argininosuccinate Synthetase 1 (ASS)-dependent amino acid pathways, resulting in AML cell death. T cell proliferation is restored in vitro and in vivo. The capacity of iNKT cells to restore antigen-specific T cell immunity was similarly demonstrated against myeloid-derived suppressor cells (MDSCs) in wild-type and Jα18−/− syngeneic lymphoma-bearing models in vivo. Thus, stimulation of iNKT cell activity has the potential as an immunotherapy against AML or as an adjunct to boost antigen-specific T cell immunotherapies in haematological or solid cancers.

Spatial separation of ions in the gas-phase, providing information about their size as collisional cross-sections, can readily be achieved through ion mobility. The timsTOF Pro series combines a trapped ion mobility device with a quadrupole, collision-cell and a time-of-flight analyser to enable the analysis of ions at great speed. Here, we show that the timsTOF Pro is capable of physically separating N-glycopeptides from non-modified peptides and producing high-quality fragmentation spectra, both beneficial for glycoproteomics analyses of complex samples. The glycan moieties enlarge the size of glycopeptides compared to non-modified peptides, yielding a clear cluster in the mobilogram that, next to increased dynamic range from the physical separation of glycopeptides and non-modified peptides, can be used to make an effective selection filter for directing the mass spectrometer to analytes of interest. This new approach was applied to selected glycoproteins, human plasma- and neutrophil-derived glycopeptides. We show that the achieved physical separation, combined with the focussing of the mass spectrometer, allows for improved extraction of information from the samples, even at shorter LC gradients of 15 min. We validated our approach on human neutrophil and plasma samples of known make-up, in which we captured the anticipated glycan heterogeneity (paucimannose, phosphomannose, high mannose, hybrid and complex glycans) from plasma and neutrophil samples at the expected abundances. As the method is compatible with off-the-shelve data acquisition routines and data analysis software, it can readily be applied by any laboratory with a timsTOF Pro and is reproducible as demonstrated by a comparison between two laboratories.

Crosslinking mass spectrometry (XL-MS) adds tremendous value to structural biology investigations. Its main strength lies in uncovering structural information in the form of distance constraints between neighboring amino acids of proteins, protein regions and complex samples, which are difficult to assess by other analytical techniques. However, although several approaches have been proposed, interpreting XL-MS data in a structural context has been cumbersome. ChimeraX has gained momentum as a flexible and widely used software package for the visualization of structural data, but is currently lacking functionalities for integration of experimental XL-MS data. Here, we introduce XMAS, a bundle that allows users to load results from several XL-MS search engines directly into ChimeraX and map the information onto protein structures. Besides automatically locating distance constraints on protein structures, XMAS offers the possibility to work with replicate experiments and/or different crosslinkers, and filter this data based on the number of replicates for which a given distance constraint was detected, thereby increasing the data quality. Additionally, we introduce the concept of self-links, which allows easy modeling of homo-dimeric interactions. Its core functionality is extended by the implementation of seamless connections to the HADDOCK suite to streamline otherwise time-consuming tasks in structural modeling pipelines. We demonstrate these key elements of the XMAS bundle by modeling crosslinking data obtained from human fibrin clots. The software is freely available from the ChimeraX toolshed, with an extensive user manual and example datasets.