Tagless LysoIP for immunoaffinity enrichment of native lysosomes from clinical samples.

Lysosomes are implicated in a wide spectrum of human diseases including monogenic lysosomal storage disorders (LSDs), age-associated neurodegeneration and cancer. Profiling lysosomal content using tag-based lysosomal immunoprecipitation (LysoTagIP) in cell and animal models has substantially moved the field forward, but studying lysosomal dysfunction in human patients remains challenging. Here, we report the development of the 'tagless LysoIP' method, designed to enable the rapid enrichment of lysosomes, via immunoprecipitation, using the endogenous integral lysosomal membrane protein TMEM192, directly from clinical samples and human cell lines (e.

Isoleucine-to-valine substitutions support cellular physiology during isoleucine deprivation.

Aminoacyl-tRNA synthetases (ARSs) couple tRNAs with their corresponding amino acids. While ARSs can bind structurally similar amino acids, extreme specificity is ensured by subsequent editing activity. Yet, we found that upon isoleucine (I) restriction, healthy fibroblasts consistently incorporated valine (V) into proteins at isoleucine codons, resulting from misacylation of tRNAIle with valine by wildtype IARS1.

mTORC1 restricts TFE3 activity by auto-regulating its presence on lysosomes.

To stimulate cell growth, the protein kinase complex mTORC1 requires intracellular amino acids for activation. Amino-acid sufficiency is relayed to mTORC1 by Rag GTPases on lysosomes, where growth factor signaling enhances mTORC1 activity via the GTPase Rheb. In the absence of amino acids, GATOR1 inactivates the Rags, resulting in lysosomal detachment and inactivation of mTORC1.

[Two children in the Netherlands with a Corynebacterium diphtheriaeinfection].

Infections with were virtually absent among the Dutch population due to vaccination, while in the first half of the 20th century, it was a significant cause of child mortality. However, due to imported infections resulting from migration from countries with low vaccination coverage, infections with are resurging. Concurrently, the vaccination rates among Dutch children are decreasing, elevating the risk of outbreaks.

Leaving no patient behind! Expert recommendation in the use of innovative technologies for diagnosing rare diseases.

Genetic diagnosis plays a crucial role in rare diseases, particularly with the increasing availability of emerging and accessible treatments. The International Rare Diseases Research Consortium (IRDiRC) has set its primary goal as: "Ensuring that all patients who present with a suspected rare disease receive a diagnosis within one year if their disorder is documented in the medical literature". Despite significant advances in genomic sequencing technologies, more than half of the patients with suspected Mendelian disorders remain undiagnosed.

Inborn errors of the malate aspartate shuttle - Update on patients and cellular models.

The malate aspartate shuttle (MAS) plays a pivotal role in transporting cytosolic reducing equivalents - electrons - into the mitochondria for energy conversion at the electron transport chain (ETC) and in the process of oxidative phosphorylation. The MAS consists of two pairs of cytosolic and mitochondrial isoenzymes (malate dehydrogenases 1 and 2; and glutamate oxaloacetate transaminases 1 and 2) and two transporters (malate-2-oxoglutarate carrier and aspartate glutamate carrier (AGC), the latter of which has two tissue-dependent isoforms AGC1 and AGC2). While the inner mitochondrial membrane is impermeable to NADH, the MAS forms one of the main routes for mitochondrial electron uptake by promoting uptake of malate.

Metabolic blood profile and response to treatment with the pyruvate kinase activator mitapivat in patients with sickle cell disease.

Mitapivat is an investigational, oral, small-molecule allosteric activator of pyruvate kinase (PK). PK is a regulatory glycolytic enzyme that is key in providing the red blood cell (RBC) with sufficient amounts of adenosine triphosphate (ATP). In sickle cell disease (SCD), decreased 2,3-DPG levels increase the oxygen affinity of hemoglobin, thereby preventing deoxygenation and polymerization of sickle hemoglobin.

Direct Infusion Mass Spectrometry to Rapidly Map Metabolic Flux of Substrates Labeled with Stable Isotopes.

Direct infusion-high-resolution mass spectrometry (DI-HRMS) allows for rapid profiling of complex mixtures of metabolites in blood, cerebrospinal fluid, tissue samples and cultured cells. Here, we present a DI-HRMS method suitable for the rapid determination of metabolic fluxes of isotopically labeled substrates in cultured cells and organoids. We adapted an automated annotation pipeline by selecting labeled adducts that best represent the majority of C and/or N-labeled glycolytic and tricarboxylic acid cycle intermediates as well as a number of their derivatives.

The monocyte-derived cytokine response in whole blood from preterm newborns against sepsis-related bacteria is similar to term newborns and adults.

Introduction: Sepsis is characterized by a dysregulated innate immune response. It is a leading cause of morbidity and mortality in newborns, in particular for newborns that are born premature. Although previous literature indicate that the pro-inflammatory response may be impaired in preterm newborns, serum levels of monocyte-derived cytokines, such as TNF-α and IL-6, vary highly between newborns and can reach adult-like concentrations during sepsis.

A novel composition of endogenous metabolic modulators improves red blood cell properties in sickle cell disease.

The most common forms of sickle cell disease (SCD) are sickle cell anemia (SCA; HbSS) and HbSC disease. In both, especially the more dense, dehydrated and adherent red blood cells (RBCs) with reduced deformability are prone to hemolysis and sickling, and thereby vaso-occlusion. Based on plasma amino acid profiling in SCD, a composition of 10 amino acids and derivatives (RCitNacQCarLKHVS; Axcella Therapeutics, USA), referred to as endogenous metabolic modulators (EMMs), was designed to target RBC metabolism.

UPLC-Orbitrap-HRMS application for analysis of plasma sterols.

Correct identification and quantification of different sterol biomarkers can be used as a first-line diagnostic approach for inherited metabolic disorders (IMD). The main drawbacks of current methodologies are related to lack of selectivity and sensitivity for some of these compounds. To address this, we developed and validated two sensitive and selective assays for quantification of six cholesterol biosynthesis pathway intermediates (total amount (free and esterified form) of 7-dehydrocholesterol (7-DHC), 8-dehydrocholesterol (8-DHC), desmosterol, lathosterol, lanosterol and cholestanol), two phytosterols (total amount (free and esterified form) of campesterol and sitosterol) and free form of two oxysterols (7-ketocholesterol (7-KC) and 3β,5α,6β-cholestane-triol (C-triol).

Metabolic Alterations in NADSYN1-Deficient Cells.

NAD synthetase 1 (encoded by the gene NADSYN1) is a cytosolic enzyme that catalyzes the final step in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) from tryptophan and nicotinic acid. NADSYN1 deficiency has recently been added to the spectrum of congenital NAD+ deficiency disorders. To gain insight into the metabolic consequences of NADSYN1 deficiency, the encoding gene was disrupted in A549 and HEK293T cells, and the metabolome was profiled in the presence of different NAD+ precursors, including tryptophan, nicotinamide and nicotinic acid.

Inflammation-induced mitochondrial and metabolic disturbances in sensory neurons control the switch from acute to chronic pain.

Pain often persists in patients with an inflammatory disease, even when inflammation has subsided. The molecular mechanisms leading to this failure in pain resolution and the transition to chronic pain are poorly understood. Mitochondrial dysfunction in sensory neurons links to chronic pain, but its role in resolution of inflammatory pain is unclear.

One-year safety and efficacy of mitapivat in sickle cell disease: follow-up results of a phase 2, open-label study.

Targeting the primary pathogenic event of sickle cell disease (SCD), the polymerization of sickle hemoglobin (HbS), may prevent downstream clinical events. Mitapivat, an oral pyruvate kinase (PK) activator, has therapeutic potential by increasing adenosine triphosphate (ATP) and decreasing 2,3-diphosphoglycerate (2,3-DPG), a glycolytic red blood cell (RBC) intermediate. In the previously reported 8-week dose-finding period of this phase 2, investigator-initiated, open-label study, mitapivat was well tolerated and showed efficacy in SCD.

Parasitic, bacterial, viral, immune-mediated, metabolic and nutritional factors associated with nodding syndrome.

Nodding syndrome is a neglected, disabling and potentially fatal epileptic disorder of unknown aetiology affecting thousands of individuals mostly confined to Eastern sub-Saharan Africa. Previous studies have identified multiple associations-including , antileiomodin-1 antibodies, vitamin B deficiency and measles virus infection-yet, none is proven causal. We conducted a case-control study of children with early-stage nodding syndrome (symptom onset <1 year).

Abnormal glucose homeostasis and fasting intolerance in patients with congenital porto-systemic shunts.

In physiological glucose homeostasis, the liver plays a crucial role in the extraction of glucose from the portal circulation and storage as glycogen to enable release through glycogenolysis upon fasting. In addition, insulin secreted by the pancreas is partly eliminated from the systemic circulation by hepatic first-pass. Therefore, patients with a congenital porto-systemic shunt present a unique combination of (a) postabsorptive hyperinsulinemic hypoglycaemia (HH) because of decreased insulin elimination and (b) fasting (ketotic) hypoglycaemia because of decreased glycogenolysis.

The malate-aspartate shuttle is important for de novo serine biosynthesis.

The malate-aspartate shuttle (MAS) is a redox shuttle that transports reducing equivalents across the inner mitochondrial membrane while recycling cytosolic NADH to NAD. We genetically disrupted each MAS component to generate a panel of MAS-deficient HEK293 cell lines in which we performed [U-C]-glucose tracing. MAS-deficient cells have reduced serine biosynthesis, which strongly correlates with the lactate M+3/pyruvate M+3 ratio (reflective of the cytosolic NAD/NADH ratio), consistent with the NAD dependency of phosphoglycerate dehydrogenase in the serine synthesis pathway.

Maintenance of cellular vitamin B levels and mitochondrial oxidative function depend on pyridoxal 5'-phosphate homeostasis protein.

Recently, biallelic variants in PLPBP coding for pyridoxal 5'-phosphate homeostasis protein (PLPHP) were identified as a novel cause of early-onset vitamin B-dependent epilepsy. The molecular function and precise role of PLPHP in vitamin B metabolism are not well understood. To address these questions, we used PLPHP-deficient patient skin fibroblasts and HEK293 cells and YBL036C (PLPHP ortholog)-deficient yeast.

Circulating Acylcarnitines Associated with Hypertrophic Cardiomyopathy Severity: an Exploratory Cross-Sectional Study in MYBPC3 Founder Variant Carriers.

Hypertrophic cardiomyopathy (HCM) is a relatively common genetic heart disease characterised by myocardial hypertrophy. HCM can cause outflow tract obstruction, sudden cardiac death and heart failure, but severity is highly variable. In this exploratory cross-sectional study, circulating acylcarnitines were assessed as potential biomarkers in 124 MYBPC3 founder variant carriers (59 with severe HCM, 26 with mild HCM and 39 phenotype-negative [G + P-]).

A second case of glutaminase hyperactivity: Expanding the phenotype with epilepsy.

Glutaminase (GLS) hyperactivity was first described in 2019 in a patient with profound developmental delay and infantile cataract. Here, we describe a 4-year-old boy with GLS hyperactivity due to a de novo heterozygous missense variant in , detected by trio whole exome sequencing. This boy also exhibits developmental delay without dysmorphic features, but does not have cataract.

Untargeted Metabolomics Identifies Potential Hypertrophic Cardiomyopathy Biomarkers in Carriers of Founder Variants.

Hypertrophic cardiomyopathy (HCM) is the most prevalent monogenic heart disease, commonly caused by pathogenic variants, and a significant cause of sudden cardiac death. Severity is highly variable, with incomplete penetrance among genotype-positive family members. Previous studies demonstrated metabolic changes in HCM.

Viral, Bacterial, Metabolic, and Autoimmune Causes of Severe Acute Encephalopathy in Sub-Saharan Africa: A Multicenter Cohort Study.

Objectives: To assess whether viral, bacterial, metabolic, and autoimmune diseases are missed by conventional diagnostics among children with severe acute encephalopathy in sub-Saharan Africa.

Study Design: One hundred thirty-four children (6 months to 18 years) presenting with nontraumatic coma or convulsive status epilepticus to 1 of 4 medical referral centers in Uganda, Malawi, and Rwanda were enrolled between 2015 and 2016. Locally available diagnostic tests could be supplemented in 117 patients by viral, bacterial, and 16s quantitative polymerase chain reaction testing, metagenomics, untargeted metabolomics, and autoimmune immunohistochemistry screening.

Multi-country metabolic signature discovery for chicken health classification.

Introduction: To decrease antibiotic resistance, their use as growth promoters in the agricultural sector has been largely abandoned. This may lead to decreased health due to infectious disease or microbiome changes leading to gut inflammation.

Objectives: We aimed to generate a m/z signature classifying chicken health in blood, and obtain biological insights from the resulting m/z signature.