Novel cerebrospinal fluid biomarkers of glucose transporter type 1 deficiency syndrome: Implications beyond the brain's energy deficit

We used next-generation metabolic screening to identify new biomarkers for improved diagnosis and pathophysiological understanding of glucose transporter type 1 deficiency syndrome (GLUT1DS), comparing metabolic cerebrospinal fluid (CSF) profiles from 12 patients to those of 116 controls. This confirmed decreased CSF glucose and lactate levels in patients with GLUT1DS and increased glutamine at group level. We identified three novel biomarkers significantly decreased in patients, namely gluconic + galactonic acid, xylose-α1-3-glucose, and xylose-α1-3-xylose-α1-3-glucose, of which the latter two have not previously been identified in body fluids. CSF concentrations of gluconic + galactonic acid may be reduced as these metabolites could serve as alternative substrates for the pentose phosphate pathway. Xylose-α1-3-glucose and xylose-α1-3-xylose-α1-3-glucose may originate from glycosylated proteins; their decreased levels are hypothetically the consequence of insufficient glucose, one of two substrates for O-glucosylation. Since many proteins are O-glucosylated, this deficiency may affect cellular processes and thus contribute to GLUT1DS pathophysiology. The novel CSF biomarkers have the potential to improve the biochemical diagnosis of GLUT1DS. Our findings imply that brain glucose deficiency in GLUT1DS may cause disruptions at the cellular level that go beyond energy metabolism, underlining the importance of developing treatment strategies that directly target cerebral glucose uptake

Initial Steps towards Spatiotemporal Signaling through Biomaterials Using Click-to-Release Chemistry

The process of wound healing is a tightly controlled cascade of events, where severe skin wounds are resolved via scar tissue. This fibrotic response may be diminished by applying anti-fibrotic factors to the wound, thereby stimulating regeneration over scarring. The development of tunable biomaterials that enable spatiotemporal control over the release of anti-fibrotics would greatly benefit wound healing. Herein, harnessing the power of click-to-release chemistry for regenerative medicine, we demonstrate the feasibility of such an approach. For this purpose, one side of a bis-N-hydroxysuccinimide-trans-cyclooctene (TCO) linker was functionalized with human epidermal growth factor (hEGF), an important regulator during wound healing, whereas on the other side a carrier protein was conjugated—either type I collagen scaffolds or bovine serum albumin (BSA). Mass spectrometry demonstrated the coupling of hEGF–TCO and indicated a release following exposure to dimethyl-tetrazine. Type I collagen scaffolds could be functionalized with the hEGF–TCO complex as demonstrated by immunofluorescence staining and Western blotting. The hEGF–TCO complex was also successfully ligated to BSA and the partial release of hEGF upon dimethyl-tetrazine exposure was observed through Western blotting. This work establishes the potential of click-to-release chemistry for the development of pro-regenerative biomaterials

Fine-tuning of lysine side chain modulates the activity of histone lysine methyltransferases

Contains fulltext : 230075.pdf (publisher's version ) (Open Access

Luminescent Assay for the Screening of SARS-CoV-2 MPro Inhibitors

Since the outbreak of SARS-CoV-2 in December 2019 millions of infections have been reported globally. The viral chymotrypsin-like main protease (MPro) exhibits a crucial role in viral replication and represents a relevant target for antiviral drug development. In order to screen potential MPro inhibitors we developed a luminescent assay using a peptide based probe containing a cleavage site specific for MPro. This assay was validated showing IC50 values similar to those reported in the literature for known MPro inhibitors and can be used to screen new inhibitors

Metabolite Identification Using Infrared Ion Spectroscopy – Novel Biomarkers for Pyridoxine-Dependent Epilepsy

Untargeted LC-MS based metabolomics strategies are being increasingly applied in metabolite screening for a wide variety of medical conditions. The long-standing “grand challenge” in the utilization of this approach is metabolite identification – confidently determining the chemical structures of m/z-detected unknowns. Here, we use a novel workflow based on the detection of molecular features of interest by high-throughput untargeted LC-MS analysis of patient body fluids combined with targeted molecular identification of those features using infrared ion spectroscopy (IRIS), effectively providing diagnostic IR fingerprints for mass-isolated targets. A significant advantage of this approach is that in silico predicted IR spectra of candidate chemical structures can be used to suggest the molecular structure of unknown features, thus mitigating the need for the synthesis of a broad range of physical reference standards. Pyridoxine dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine metabolism, resulting from a mutation in the ALDH7A1 gene that leads to an accumulation of toxic levels of α-aminoadipic semialdehyde (α-AASA), piperideine-6-carboxylate (P6C), and pipecolic acid in body fluids. While α-AASA and P6C are known biomarkers for PDE in urine, their instability makes them poor candidates for diagnostic analysis from blood, which would be required for application in newborn screening protocols. Here, we use combined untargeted metabolomics-IRIS to identify several new biomarkers for PDE-ALDH7A1 that can be used for diagnostic analysis in urine, plasma, and cerebrospinal fluids, and are compatible with analysis in dried blood spots for newborn screening. The identification of these novel metabolites has directly rendered novel insights in the pathophysiology of PDE-ALDH7A1

Identification of Δ-1-pyrroline-5-carboxylate derived biomarkers for hyperprolinemia type II

Hyperprolinemia type II (HPII) is an inborn error of metabolism due to genetic variants in ALDH4A1, leading to a deficiency in Δ-1-pyrroline-5-carboxylate (P5C) dehydrogenase. This leads to an accumulation of toxic levels of P5C, an intermediate in proline catabolism. The accumulating P5C spontaneously reacts with, and inactivates, pyridoxal 5'-phosphate, a crucial cofactor for many enzymatic processes, which is thought to be the pathophysiological mechanism for HPII. Here, we describe the use of a combination of LC-QTOF untargeted metabolomics, NMR spectroscopy and infrared ion spectroscopy (IRIS) to identify and characterize biomarkers for HPII that result of the spontaneous reaction of P5C with malonic acid and acetoacetic acid. We show that these biomarkers can differentiate between HPI, caused by a deficiency of proline oxidase activity, and HPII. The elucidation of their molecular structures yields insights into the disease pathophysiology of HPII

Metabolite Identification Using Infrared Ion Spectroscopy-Novel Biomarkers for Pyridoxine-Dependent Epilepsy

Untargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics strategies are being increasingly applied in metabolite screening for a wide variety of medical conditions. The long-standing grand challenge in the utilization of this approach is metabolite identification-confidently determining the chemical structures of m/z-detected unknowns. Here, we use a novel workflow based on the detection of molecular features of interest by high-throughput untargeted LC-MS analysis of patient body fluids combined with targeted molecular identification of those features using infrared ion spectroscopy (IRIS), effectively providing diagnostic IR fingerprints for mass-isolated targets. A significant advantage of this approach is that in silico-predicted IR spectra of candidate chemical structures can be used to suggest the molecular structure of unknown features, thus mitigating the need for the synthesis of a broad range of physical reference standards. Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine metabolism, resulting from a mutation in the ALDH7A1 gene that leads to an accumulation of toxic levels of alpha-aminoadipic semialdehyde (alpha-AASA), piperideine-6-carboxylate (P6C), and pipecolic acid in body fluids. While alpha-AASA and P6C are known biomarkers for PDE in urine, their instability makes them poor candidates for diagnostic analysis from blood, which would be required for application in newborn screening protocols. Here, we use combined untargeted metabolomics-IRIS to identify several new biomarkers for PDE-ALDH7A1 that can be used for diagnostic analysis in urine, plasma, and cerebrospinal fluids and that are compatible with analysis in dried blood spots for newborn screening. The identification of these novel metabolites has directly provided novel insights into the pathophysiology of PDE-ALDH7A1

Metabolite Identification Using Infrared Ion Spectroscopy-Novel Biomarkers for Pyridoxine-Dependent Epilepsy

Untargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics strategies are being increasingly applied in metabolite screening for a wide variety of medical conditions. The long-standing "grand challenge"in the utilization of this approach is metabolite identification-confidently determining the chemical structures of m/z-detected unknowns. Here, we use a novel workflow based on the detection of molecular features of interest by high-throughput untargeted LC-MS analysis of patient body fluids combined with targeted molecular identification of those features using infrared ion spectroscopy (IRIS), effectively providing diagnostic IR fingerprints for mass-isolated targets. A significant advantage of this approach is that in silico-predicted IR spectra of candidate chemical structures can be used to suggest the molecular structure of unknown features, thus mitigating the need for the synthesis of a broad range of physical reference standards. Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine metabolism, resulting from a mutation in the ALDH7A1 gene that leads to an accumulation of toxic levels of α-aminoadipic semialdehyde (α-AASA), piperideine-6-carboxylate (P6C), and pipecolic acid in body fluids. While α-AASA and P6C are known biomarkers for PDE in urine, their instability makes them poor candidates for diagnostic analysis from blood, which would be required for application in newborn screening protocols. Here, we use combined untargeted metabolomics-IRIS to identify several new biomarkers for PDE-ALDH7A1 that can be used for diagnostic analysis in urine, plasma, and cerebrospinal fluids and that are compatible with analysis in dried blood spots for newborn screening. The identification of these novel metabolites has directly provided novel insights into the pathophysiology of PDE-ALDH7A1

Untargeted metabolomics and infrared ion spectroscopy identify biomarkers for pyridoxine-dependent epilepsy

Background. Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine catabolism that presents with refractory epilepsy in newborns. Biallelic ALDH7A1 variants lead to deficiency of α-aminoadipic semialdehyde dehydrogenase/ antiquitin, resulting in accumulation of piperideine-6-carboxylate (P6C), and secondary deficiency of the important cofactor pyridoxal-5′-phosphate (PLP, active vitamin B6) through its complexation with P6C. Vitamin B6 supplementation resolves epilepsy in patients, but intellectual disability may still develop. Early diagnosis and treatment, preferably based on newborn screening, could optimize long-term clinical outcome. However, no suitable PDE-ALDH7A1 newborn screening biomarkers are currently available. Methods. We combined the innovative analytical methods untargeted metabolomics and infrared ion spectroscopy to discover and identify biomarkers in plasma that would allow for PDE-ALDH7A1 diagnosis in newborn screening. Results. We identified 2S,6S-/2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) as a PDE-ALDH7A1 biomarker, and confirmed 6-oxopiperidine-2-carboxylic acid (6-oxoPIP) as a biomarker. The suitability of 2-OPP as a potential PDE-ALDH7A1 newborn screening biomarker in dried bloodspots was shown. Additionally, we found that 2-OPP accumulates in brain tissue of patients and Aldh7a1-knockout mice, and induced epilepsy-like behavior in a zebrafish model system. Conclusion. This study has opened the way to newborn screening for PDE-ALDH7A1. We speculate that 2-OPP may contribute to ongoing neurotoxicity, also in treated PDE-ALDH7A1 patients. As 2-OPP formation appears to increase upon ketosis, we emphasize the importance of avoiding catabolism in PDE-ALDH7A1 patients

Untargeted metabolomics and infrared ion spectroscopy identify biomarkers for pyridoxine-dependent epilepsy

Background. Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine catabolism that presents with refractory epilepsy in newborns. Biallelic ALDH7A1 variants lead to deficiency of α-aminoadipic semialdehyde dehydrogenase/ antiquitin, resulting in accumulation of piperideine-6-carboxylate (P6C), and secondary deficiency of the important cofactor pyridoxal-5′-phosphate (PLP, active vitamin B6) through its complexation with P6C. Vitamin B6 supplementation resolves epilepsy in patients, but intellectual disability may still develop. Early diagnosis and treatment, preferably based on newborn screening, could optimize long-term clinical outcome. However, no suitable PDE-ALDH7A1 newborn screening biomarkers are currently available. Methods. We combined the innovative analytical methods untargeted metabolomics and infrared ion spectroscopy to discover and identify biomarkers in plasma that would allow for PDE-ALDH7A1 diagnosis in newborn screening. Results. We identified 2S,6S-/2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) as a PDE-ALDH7A1 biomarker, and confirmed 6-oxopiperidine-2-carboxylic acid (6-oxoPIP) as a biomarker. The suitability of 2-OPP as a potential PDE-ALDH7A1 newborn screening biomarker in dried bloodspots was shown. Additionally, we found that 2-OPP accumulates in brain tissue of patients and Aldh7a1-knockout mice, and induced epilepsy-like behavior in a zebrafish model system. Conclusion. This study has opened the way to newborn screening for PDE-ALDH7A1. We speculate that 2-OPP may contribute to ongoing neurotoxicity, also in treated PDE-ALDH7A1 patients. As 2-OPP formation appears to increase upon ketosis, we emphasize the importance of avoiding catabolism in PDE-ALDH7A1 patients