О нижней оценке для одной квадратичной задачи намногообразии Штифеля

Despite technological advances in metabolomics, large parts of the human metabolome are still unexplored. In an untargeted metabolomics screen aiming to identify substrates of the orphan transporter ATP-binding cassette subfamily C member 5 (ABCC5), we identified a class of mammalian metabolites, N-lactoyl-amino acids. Using parallel protein fractionation in conjunction with shotgun proteomics on fractions containing N-lactoyl-Phe-forming activity, we unexpectedly found that a protease, cytosolic nonspecific dipeptidase 2 (CNDP2), catalyzes their formation. N-lactoyl-amino acids are ubiquitous pseudodipeptides of lactic acid and amino acids that are rapidly formed by reverse proteolysis, a process previously considered to be negligible in vivo. The plasma levels of these metabolites strongly correlate with plasma levels of lactate and amino acid, as shown by increased levels after physical exercise and in patients with phenylketonuria who suffer from elevated Phe levels. Our approach to identify unknown metabolites and their biosynthesis has general applicability in the further exploration of the human metabolome

Impact of chemistry and nanoformulation parameters on cellular uptake and airway distribution of RNA oligonucleotides

Small, synthetic oligonucleotides (ON) are of great interest as potential disease modifying drugs, mainly because of their ability to modulate previously undruggable target mutations. To date, therapeutic applications of ON are, however, limited by their physicochemical properties, including poor stability, rapid excretion and low intracellular access. In order to overcome some of these shortcomings, ON are generally formulated using nanoparticle (NP) delivery systems. Alternatively, the poor stability can be circumvented by including chemical modifications to the backbone or sugars of the ON. Some of these modifications also result in better intracellular target access of these otherwise membrane-impermeable macromolecules. Therefore, complex formulation of ON into NP in order to overcome the hurdle of intracellular access might not always be needed, especially in case of local delivery. In this study, the delivery and functionality of chemically modified ON in free form was compared to polymeric NP assisted delivery, measuring their effectivity and efficiency. For this reason, phosphorothioate (PS) backbone-modified 18-mer ON with either 2'OMe or 2'MOE-modifications were selected, capable of eliciting exon-skipping of an aberrant exon in fluorescence based in vitro and in vivo model systems. The NP consisted of poly(D,L-lactic,co-glycolic acid) and poly-β-amino-ester, previously demonstrated to successfully deliver nucleic acids via the pulmonary route. Several NP formulation parameters were tested in order to optimize the delivery of the ON, including ratio polymer:ON, NP size and concentration. The results reported here show clear differences between gymnotic and nanoparticle mediated ON delivery in terms of cellular uptake and local tissue distribution. In vitro, differences in exon-skipping efficiencies were observed with 2'OMe and 2'MOE ON either in free form or formulated in NP, with the striking observation that 2'OMe ON formulated in polymeric NP did not result in exon skipping. Gymnotic delivery of 2'MOE ON into the respiratory tract of mice resulted in functional delivery of exon-skipping ON into nasal epithelia and lungs as well as other downstream tissues and organs, pointing towards a gradual redistribution of locally delivered ONs, with limited but measurable systemic exposure. Conversely, NP-mediated delivery into the respiratory tract resulted in a more contained functional delivery at 10× lower ON doses compared to gymnotic delivery. Based on these findings we conclude that gymnotic delivery of 2'OMe or 2'MOE exon-skipping ON to the respiratory tract is effective, but that NP formulation might be advantageous in case spread of ON to non-target tissue can lead to undesired effects

Impact of chemistry and nanoformulation parameters on cellular uptake and airway distribution of RNA oligonucleotides

Small, synthetic oligonucleotides (ON) are of great interest as potential disease modifying drugs, mainly because of their ability to modulate previously undruggable target mutations. To date, therapeutic applications of ON are, however, limited by their physicochemical properties, including poor stability, rapid excretion and low intracellular access. In order to overcome some of these shortcomings, ON are generally formulated using nanoparticle (NP) delivery systems. Alternatively, the poor stability can be circumvented by including chemical modifications to the backbone or sugars of the ON. Some of these modifications also result in better intracellular target access of these otherwise membrane-impermeable macromolecules. Therefore, complex formulation of ON into NP in order to overcome the hurdle of intracellular access might not always be needed, especially in case of local delivery. In this study, the delivery and functionality of chemically modified ON in free form was compared to polymeric NP assisted delivery, measuring their effectivity and efficiency. For this reason, phosphorothioate (PS) backbone-modified 18-mer ON with either 2'OMe or 2'MOE-modifications were selected, capable of eliciting exon-skipping of an aberrant exon in fluorescence based in vitro and in vivo model systems. The NP consisted of poly(D,L-lactic,co-glycolic acid) and poly-β-amino-ester, previously demonstrated to successfully deliver nucleic acids via the pulmonary route. Several NP formulation parameters were tested in order to optimize the delivery of the ON, including ratio polymer:ON, NP size and concentration. The results reported here show clear differences between gymnotic and nanoparticle mediated ON delivery in terms of cellular uptake and local tissue distribution. In vitro, differences in exon-skipping efficiencies were observed with 2'OMe and 2'MOE ON either in free form or formulated in NP, with the striking observation that 2'OMe ON formulated in polymeric NP did not result in exon skipping. Gymnotic delivery of 2'MOE ON into the respiratory tract of mice resulted in functional delivery of exon-skipping ON into nasal epithelia and lungs as well as other downstream tissues and organs, pointing towards a gradual redistribution of locally delivered ONs, with limited but measurable systemic exposure. Conversely, NP-mediated delivery into the respiratory tract resulted in a more contained functional delivery at 10× lower ON doses compared to gymnotic delivery. Based on these findings we conclude that gymnotic delivery of 2'OMe or 2'MOE exon-skipping ON to the respiratory tract is effective, but that NP formulation might be advantageous in case spread of ON to non-target tissue can lead to undesired effects

ABCC6-mediated ATP secretion by the liver is the main source of the mineralization inhibitor inorganic pyrophosphate in the systemic circulation-brief report

Mutations in ABCC6 underlie the ectopic mineralization disorder pseudoxanthoma elasticum (PXE) and some forms of generalized arterial calcification of infancy, both of which affect the cardiovascular system. Using cultured cells, we recently showed that ATP-binding cassette subfamily C member 6 (ABCC6) mediates the cellular release of ATP, which is extracellularly rapidly converted into AMP and the mineralization inhibitor inorganic pyrophosphate (PPi). The current study was performed to determine which tissues release ATP in an ABCC6-dependent manner in vivo, where released ATP is converted into AMP and PPi, and whether human PXE ptients have low plasma PPi concentrations. Using cultured primary hepatocytes and in vivo liver perfusion experiments, we found that ABCC6 mediates the direct, sinusoidal, release of ATP from the liver. Outside hepatocytes, but still within the liver vasculature, released ATP is converted into AMP and PPi. The absence of functional ABCC6 in patients with PXE leads to strongly reduced plasma PPi concentrations. Hepatic ABCC6-mediated ATP release is the main source of circulating PPi, revealing an unanticipated role of the liver in systemic PPi homeostasis. Patients with PXE have a strongly reduced plasma PPi level, explaining their mineralization disorder. Our results indicate that systemic PPi is relatively stable and that PXE, generalized arterial calcification of infancy, and other ectopic mineralization disorders could be treated with PPi supplementation therap