Localized mixed-valence and redox activity within a triazole-bridged dinucleating ligand upon coordination to palladium

The new dinucleating redox-active ligand (LH4), bearing two redox-active NNO-binding pockets linked by a 1,2,3-triazole unit, is synthetically readily accessible. Coordination to two equivalents of PdII resulted in the formation of paramagnetic (S=inline image ) dinuclear Pd complexes with a κ2-N,N′-bridging triazole and a single bridging chlorido or azido ligand. A combined spectroscopic, spectroelectrochemical, and computational study confirmed Robin–Day Class II mixed-valence within the redox-active ligand, with little influence of the secondary bridging anionic ligand. Intervalence charge transfer was observed between the two ligand binding pockets. Selective one-electron oxidation allowed for isolation of the corresponding cationic ligand-based diradical species. SQUID (super-conducting quantum interference device) measurements of these compounds revealed weak anti-ferromagnetic spin coupling between the two ligand-centered radicals and an overall singlet ground state in the solid state, which is supported by DFT calculations. The rigid and conjugated dinucleating redox-active ligand framework thus allows for efficient electronic communication between the two binding pockets

Inter-laboratory analytical improvement of succinylacetone and nitisinone quantification from dried blood spot samples

Background: Nitisinone is used to treat hereditary tyrosinemia type 1 (HT-1) by preventing accumulation of toxic metabolites, including succinylacetone (SA). Accurate quantification of SA during newborn screening is essential, as is quantification of both SA and nitisinone for disease monitoring and optimization of treatment. Analysis of dried blood spots (DBS) rather than plasma samples is a convenient method, but interlaboratory differences and comparability of DBS to serum/plasma may be issues to consider. Methods: Eight laboratories with experience in newborn screening and/or monitoring of patients with HT-1 across Europe participated in this study to assess variability and improve SA and nitisinone concentration measurements from DBS by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Quantification of nitisinone from both DBS and plasma was performed to assess sample comparability. In addition, efforts to harmonize laboratoryprocedures of SA and nitisinone quantifications during 5 rounds of analysis are described. Results: Nitisinone levels measured from DBS and plasma strongly correlated (R2 = 0.93). Due to partitioning of nitisinone to the plasma, levels were higher in plasma by a factor of 2.34. In the initial assessment of laboratory performance, all had linear calibrations of SA and nitisinone although there was large inter-laboratory variability in actual concentration measurements. Subsequent analytical rounds demonstrated markedly improved spread and precision over previous rounds, an outcome confirmed in a final re-test round. Conclusion: The study provides guidance for the determination of nitisinone and SA from DBS and the interpretation of results in the clinic. Interlaboratory analytical harmonization was demonstrated through calibration improvements.SCOPUS: ar.kinfo:eu-repo/semantics/publishe

Myoglobin expression improves T-cell metabolism and antitumor effector function

Background The tumor microenvironment is frequently hypoxic and characterized by a scarcity of nutritional resources including a shortage of glucose. As effector T cells have high energy demands, tumor metabolism can contribute to T-cell dysfunction and exhaustion. Methods In this study, we determined hypoxia in spleen and tumor tissue from tumor-bearing C57BL/6J mice using reverse transcription polymerase chain reaction (RT-PCR), histology and flow cytometry. Next, CD8 + T cells isolated from C57BL6J mice or P14 + mice were transduced with Thy1.1 (Control) or Thy1.1-Myoglobin (Mb) packaged retrovirus. Expression of Mb was confirmed with RT-PCR and western blot. Cellular metabolism was determined by flow cytometry, transmission electron microscopy, focused ion beam scanning electron microscopy, Seahorse, metabolomics and luminescence assays. Mb expressing or control P14 + or OT-I + T cells were transferred in B16F10-gp33 or MC38-ova tumor-bearing mice respectively and analyzed using flow cytometry and histology. B16F10-gp33 tumor-bearing mice were additionally treated with anti-programmed cell death protein-1 (PD-1) checkpoint inhibitor. Results Here we demonstrate that expression of the oxygen-binding protein myoglobin in T cells can boost their mitochondrial and glycolytic metabolic functions. Metabolites and tricarboxylic acid compounds were highly increased in the presence of myoglobin (Mb), which was associated with increased ATP levels. Mb-expressing T cells exhibited low expression of hypoxia-inducible factor-1α after activation and during infiltration into the tumor microenvironment (TME). Accordingly, Mb expression increased effector T-cell function against tumor cells in vitro with concomitant reductions in superoxide levels. Following adoptive transfer into tumor-bearing mice, Mb expression facilitated increased infiltration into the TME. Although T cells expressing Mb exhibited increased expression of effector cytokines, PD-1 was still detected and targetable by anti-PD-1 monoclonal antibodies, which in combination with transfer of Mb-expressing T cells demonstrated maximal efficacy in delaying tumor growth. Conclusion Taken together, we show that expression of Mb in T cells can increase their metabolism, infiltration into the tumor tissue, and effector function against cancer cells.</p

Cardiometabolic risk factor clustering in patients with deficient branched‐chain amino acid catabolism: A case‐control study

AbstractClassical organic acidemias (OAs) result from defective mitochondrial catabolism of branched‐chain amino acids (BCAAs). Abnormal mitochondrial function relates to oxidative stress, ectopic lipids and insulin resistance (IR). We investigated whether genetically impaired function of mitochondrial BCAA catabolism associates with cardiometabolic risk factors, altered liver and muscle energy metabolism, and IR. In this case‐control study, 31 children and young adults with propionic acidemia (PA), methylmalonic acidemia (MMA) or isovaleric acidemia (IVA) were compared with 30 healthy young humans using comprehensive metabolic phenotyping including in vivo 31P/1H magnetic resonance spectroscopy of liver and skeletal muscle. Among all OAs, patients with PA exhibited abdominal adiposity, IR, fasting hyperglycaemia and hypertriglyceridemia as well as increased liver fat accumulation, despite dietary energy intake within recommendations for age and sex. In contrast, patients with MMA more frequently featured higher energy intake than recommended and had a different phenotype including hepatomegaly and mildly lower skeletal muscle ATP content. In skeletal muscle of patients with PA, slightly lower inorganic phosphate levels were found. However, hepatic ATP and inorganic phosphate concentrations were not different between all OA patients and controls. In patients with IVA, no abnormalities were detected. Impaired BCAA catabolism in PA, but not in MMA or IVA, was associated with a previously unrecognised, metabolic syndrome‐like phenotype with abdominal adiposity potentially resulting from ectopic lipid storage. These findings suggest the need for early cardiometabolic risk factor screening in PA

Controlling mitochondrial membrane architecture via MIC60 determines viral replication to promote anti-viral immunity

Virus-infected cells often exhibit dramatic cellular changes accompanied by altered mitochondrial function. The contribution of factors shaping the inner mitochondrial membrane (IMM) and cristae architecture to viral replication is insufficiently understood. Single-cell transcriptomics applying vesicular stomatitis virus infection suggests involvement of factors determining IMM architecture following infection. Consistently, inhibition of the F1FO adenosine triphosphate (ATP) synthase reduces viral replication, which is associated with oligomerization of this complex and altered IMM structure. Moreover, deletion of mitochondrial contact site and cristae organizing system (MICOS) complex by targeting MIC60 results in reduced viral replication. Generation of Mic60inv/inv CD11c-Cre+ mice uncovers reduced crista junctions and viral replication in bone marrow-derived dendritic cells. Consequently, reduced viral replication in CD11c-expressing cells limits prolonged immune activation. Altogether, by linking the F1FO ATP synthase and the MICOS complex to viral replication and immune activation, we describe links between the mitochondrial structure-metabolism and the immune response against viral infection.</p

The role of LSR gene variants in early onset intrahepatic cholestasis: a case series with treatment options

We report on three children with novel variants in the lipolysis-stimulated lipoprotein receptor (LSR) gene with clinical presentation with early onset intrahepatic cholestasis and the main symptom being uncontrollable itching. Two patients showed dystrophy, short stature and microcephaly, whilst one patient had neurological developmental delay. LSR is one component of special tricellular tight junctions (tTJs) with expression in the liver and brain. We analyzed clinical data for all patients and performed multigene panel sequencing followed by Human Phenotype Ontology (HPO) based exome analysis, classifying the sequenced variants according to the American College of Medical Genetics and Genomics (ACMG) guidelines. We performed immunostaining on the liver cryosections. The lack of LSR expression in immunofluorescence of the patients’ liver tissue confirmed the pathogenicity of genetic variants. We analyzed bile acids (BA) and their derivatives by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) in two of the three patients, confirming disturbed bile salt secretion. We also describe the use of an ileal bile acid transport (IBAT) inhibitor in two patients with LSR-associated intrahepatic cholestasis for the first time. Both patients showed a good response to the therapy in terms of itch control. In conclusion, LSR-associated early onset intrahepatic cholestasis is a new and likely underdiagnosed disease. Patients with an unclear progressive familial intrahepatic cholestasis (PFIC)-like clinical picture should therefore undergo genetic testing of the LSR gene. Treatment with an IBAT inhibitor should be considered

A Novel Tandem Mass Spectrometry Method for Rapid Confirmation of Medium- and Very Long-Chain acyl-CoA Dehydrogenase Deficiency in Newborns

BACKGROUND:Newborn screening for medium- and very long-chain acyl-CoA dehydrogenase (MCAD and VLCAD, respectively) deficiency, using acylcarnitine profiling with tandem mass spectrometry, has increased the number of patients with fatty acid oxidation disorders due to the identification of additional milder, and so far silent, phenotypes. However, especially for VLCADD, the acylcarnitine profile can not constitute the sole parameter in order to reliably confirm disease. Therefore, we developed a new liquid chromatography tandem mass spectrometry (LC-MS/MS) method to rapidly determine both MCAD- and/or VLCAD-activity in human lymphocytes in order to confirm diagnosis. METHODOLOGY:LC-MS/MS was used to measure MCAD- or VLCAD-catalyzed production of enoyl-CoA and hydroxyacyl-CoA, in human lymphocytes. PRINCIPAL FINDINGS:VLCAD activity in controls was 6.95+/-0.42 mU/mg (range 1.95 to 11.91 mU/mg). Residual VLCAD activity of 4 patients with confirmed VLCAD-deficiency was between 0.3 and 1.1%. Heterozygous ACADVL mutation carriers showed residual VLCAD activities of 23.7 to 54.2%. MCAD activity in controls was 2.38+/-0.18 mU/mg. In total, 28 patients with suspected MCAD-deficiency were assayed. Nearly all patients with residual MCAD activities below 2.5% were homozygous 985A>G carriers. MCAD-deficient patients with one other than the 985A>G mutation had higher MCAD residual activities, ranging from 5.7 to 13.9%. All patients with the 199T>C mutation had residual activities above 10%. CONCLUSIONS:Our newly developed LC-MS/MS method is able to provide ample sensitivity to correctly and rapidly determine MCAD and VLCAD residual activity in human lymphocytes. Importantly, based on measured MCAD residual activities in correlation with genotype, new insights were obtained on the expected clinical phenotype