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The brain-related phenotypes observed in 22q11.2 deletion syndrome (DS) patients are highly variable, and their origin is poorly understood. Changes in brain metabolism might contribute to these phenotypes, as many of the deleted genes are involved in metabolic processes, but this is unknown. This study shows for the first time that Tbx1 haploinsufficiency causes brain metabolic imbalance. We studied two mouse models of 22q11.2DS using mass spectrometry, nuclear magnetic resonance spectroscopy, and transcriptomics. We found that Tbx1 +/- mice and Df1/ + mice, with a multigenic deletion that includes Tbx1 , have elevated brain methylmalonic acid, which is highly brain-toxic. Focusing on Tbx1 mutants, we found that they also have a more general brain metabolomic imbalance that affects key metabolic pathways, such as glutamine-glutamate and fatty acid metabolism. We provide transcriptomic evidence of a genotype-vitamin B12 treatment interaction. In addition, vitamin B12 treatment rescued a behavioural anomaly in Tbx1 +/- mice. Further studies will be required to establish whether the specific metabolites affected by Tbx1 haploinsufficiency are potential biomarkers of brain disease status in 22q11.2DS patients., (© 2024 Caterino et al.)

Glycogen storage disease type Ia (GSDIa) is a rare, inherited glucose-6-phosphatase-α (G6Pase-α) deficiency-induced carbohydrate metabolism disorder. Although hyperlipidemia is a hallmark of GSDI, the extent of lipid metabolism disruption remains incompletely understood. Lipidomic analysis was performed to characterize the serum lipidome in patients with GSDIa, by including age- and sex-matched healthy controls and age-matched hypercholesterolemic controls. Metabolic control and dietary information biochemical markers were obtained from patients with GSDIa. Patients with GSDIa showed higher total serum lysophosphatidylcholine (Fold Change, (FC) 2.2, P < 0.0001), acyl-acyl-phosphatidylcholine (FC 2.1, P < 0.0001), and ceramide (FC 2.4, P < 0.0001) levels and bile acid (FC 0.7, P < 0.001), acylcarnitines (FC 0.7, P < 0.001), and cholesterol esters (FC 1.0, P < 0.001) than those of healthy controls, and higher di- (FC 1.1, P < 0.0001; FC 0.9, P < 0.01) and triacylglycerol (FC 6.3, P < 0.0001; FC 3.9, P < 0.01) levels than those of healthy controls and hypercholesterolemic subjects. Both total cholesterol and triglyceride values correlated with Cer (d16:1/22:0), Cer (d18:1/20:0), Cer (d18:1/20:0(OH)), Cer (d18:1/22:0), Cer (d18:1/23:0), Cer (d18:1/24:1), Cer (d18:2/22:0), Cer (d18:2/24:1). Total cholesterol also correlated with Cer (d18:1/24:0), Cer (d18:2/20:0), HexCer (d16:1/22:0), HexCer (d18:1/18:0), and Hex2Cer (d18:1/20:0). Triglyceridelevels correlated with Cer (d18:0/24:1). Alanine aminotransferase values correlated with Cer (d18:0/22:0), insulin with Cer (d18:1/22:1) and Cer (d18:1/24:1), and HDL with hexosylceramide (HexCer) (d18:2/23:0). These results expand on the currently known involvement of lipid metabolism in GSDIa. Circulating Cer may allow for refined dietary assessment compared with traditional biomarkers. Because specific lipid species are relatively easy to assess, they represent potential novel biomarkers of GSDIa., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Objectives: Fibrosis contributes to 45% of deaths in industrialized nations and is characterized by an abnormal accumulation of extracellular matrix (ECM). There are no specific anti-fibrotic treatments for liver fibrosis, and previous unsuccessful attempts at drug development have focused on preventing ECM deposition. Because liver fibrosis is largely acknowledged to be reversible, regulating fibrosis resolution could offer novel therapeutical options. However, little is known about the mechanisms controlling ECM remodeling during resolution. Changes in proteolytic activity are essential for ECM homeostasis and macrophages are an important source of proteases. Herein, in this study we evaluate the role of macrophage-derived cathepsin D (CtsD) during liver fibrosis., Methods: CtsD expression and associated pathways were characterized in single-cell RNA sequencing and transcriptomic datasets in human cirrhosis. Liver fibrosis progression, reversion and functional characterization were assessed in novel myeloid-CtsD and hepatocyte-CtsD knock-out mice., Results: Analysis of single-cell RNA sequencing datasets demonstrated CtsD was expressed in macrophages and hepatocytes in human cirrhosis. Liver fibrosis progression, reversion and functional characterization were assessed in novel myeloid-CtsD (CtsD ΔMyel ) and hepatocyte-CtsD knock-out mice. CtsD deletion in macrophages, but not in hepatocytes, resulted in enhanced liver fibrosis. Both inflammatory and matrisome proteomic signatures were enriched in fibrotic CtsD ΔMyel livers. Besides, CtsD ΔMyel liver macrophages displayed functional, phenotypical and secretomic changes, which resulted in a degradomic phenotypical shift, responsible for the defective proteolytic processing of collagen I in vitro and impaired collagen remodeling during fibrosis resolution in vivo. Finally, CtsD-expressing mononuclear phagocytes of cirrhotic human livers were enriched in lysosomal and ECM degradative signaling pathways., Conclusions: Our work describes for the first-time CtsD-driven lysosomal activity as a central hub for restorative macrophage function during fibrosis resolution and opens new avenues to explore their degradome landscape to inform drug development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

3-methylcrotonyl-CoA carboxylase deficiency (3MCCD) is a hereditary disorder of leucine catabolism caused by pathogenetic variants in the MCCC1 or MCCC2 genes. Typically diagnosed through newborn screening (NBS), 3MCCD is characterized by elevation of 3-hydroxyisovalerylcarnitine (C5OH) in blood as well as increased excretion of 3-methylcrotonylglycine (3-MCG) in urine. While most diagnosed children remain asymptomatic, data on adults are scarce. To date, only 39 molecularly confirmed adult individuals have been reported, all being mothers diagnosed subsequent to their child NBS results. Herein, we present a 36-year-old asymptomatic man who was incidentally diagnosed with 3MCCD following his son NBS recall. Molecular analysis revealed compound heterozygosity for two pathogenic variants in the MCCC1 gene. This is the first molecularly confirmed adult man with 3MCCD reported. This case highlights the need for additional longitudinal follow-up data on individuals with 3MCCD to clarify the clinical significance of this condition and guide clinical practice, including NBS strategy., Competing Interests: The authors declare that there is no potential conflict of interest related to this work., (© 2024 The Authors. Published by Elsevier Inc.)

Background: Methylmalonic acidemia (MMA) is a rare inborn error of propionate metabolism caused by deficiency of the mitochondrial methylmalonyl-CoA mutase (MUT) enzyme. As matter of fact, MMA patients manifest impairment of the primary metabolic network with profound damages that involve several cell components, many of which have not been discovered yet. We employed cellular models and patients-derived fibroblasts to refine and uncover new pathologic mechanisms connected with MUT deficiency through the combination of multi-proteomics and bioinformatics approaches., Results: Our data show that MUT deficiency is connected with profound proteome dysregulations, revealing molecular actors involved in lysosome and autophagy functioning. To elucidate the effects of defective MUT on lysosomal and autophagy regulation, we analyzed the morphology and functionality of MMA-lysosomes that showed deep alterations, thus corroborating omics data. Lysosomes of MMA cells present as enlarged vacuoles with low degradative capabilities. Notwithstanding, treatment with an anti-propionigenic drug is capable of totally rescuing lysosomal morphology and functional activity in MUT-deficient cells. These results indicate a strict connection between MUT deficiency and lysosomal-autophagy dysfunction, providing promising therapeutic perspectives for MMA., Conclusions: Defective homeostatic mechanisms in the regulation of autophagy and lysosome functions have been demonstrated in MUT-deficient cells. Our data prove that MMA triggers such dysfunctions impacting on autophagosome-lysosome fusion and lysosomal activity., (© 2024. The Author(s).)

Mucopolysaccharidoses (MPSs) are lysosomal disorders with neurological involvement for which no cure exists. Here, we show that recombinant NK1 fragment of hepatocyte growth factor rescues substrate accumulation and lysosomal defects in MPS I, IIIA and IIIB patient fibroblasts. We investigated PI3K/Akt pathway, which is of crucial importance for neuronal function and survival, and demonstrate that PI3K inhibition abolishes NK1 therapeutic effects. We identified that autophagy inhibition, by Beclin1 silencing, reduces MPS IIIB phenotype and that NK1 downregulates autophagic-lysosome (ALP) gene expression, suggesting a possible contribution of autophagosome biogenesis in MPS. Indeed, metabolomic analyses revealed defects of mitochondrial activity accompanied by anaerobic metabolism and inhibition of AMP-activated protein kinase (AMPK), which acts on metabolism and autophagy, rescues lysosomal defects. These results provide insights into the molecular mechanisms of MPS IIIB physiopathology, supporting the development of new promising approaches based on autophagy inhibition and metabolic rewiring to correct lysosomal pathology in MPSs., Competing Interests: Luigi Michele Pavone has licensed compositions comprising hepatocyte growth factor or variants thereof for use in the treatment of mucopolysaccharidoses (granted Italian patent MI2014A001454)., (© 2024 The Author(s).)

Urea cycle disorders (UCDs) are a group of rare inborn errors of metabolism caused by a deficiency in one of the six enzymes or one of the two transporters involved in the urea cycle. Current guidelines suggest that early diagnosis and treatment of mild UCDs may improve survival and prevent decompensation and neurocognitive impairment. Nevertheless, clinical studies are very difficult to carry out in this setting due to the rarity of the diseases, and high-level evidence is scant and insufficient to draw conclusions and provide clinical guidelines. With the early introduction of newborn screening, the Italian healthcare organization fostered an advancement in expertise in metabolic disease management and screening programs, by allocating resources, and favoring the expansion of newborn screening. A group of experts operating in Italian centers decided to share their experience and provide advice for the management of mild UCDs in clinical practice. A consensus was reached by the Estimate-Talk-Estimate (ETE) method. Five items were identified, and statements for each item were agreed. Briefly, the panel advised completing the diagnosis by expanded newborn screening (ENS) with biochemical and genetic confirmation and by following up with the patient during the first year of life, with a routine laboratory and metabolic profile as well as with clinical observation. Early initiation of therapy is advised and should be followed by therapy adjustment once the diagnostic profile is completed. The therapy should be based on a low-protein diet and nitrogen scavengers. The long-term follow-up is based on growth and nutritional assessment, clinical and neurocognitive evaluation, and laboratory and instrumental parameter monitoring.

β 2 -microglobulin (β 2 -m) is a plasma protein derived from physiological shedding of the class I major histocompatibility complex (MHCI), causing human systemic amyloidosis either due to persistently high concentrations of the wild-type (WT) protein in hemodialyzed patients, or in presence of mutations, such as D76N β 2 -m, which favor protein deposition in the adulthood, despite normal plasma levels. Here we describe a new transgenic Caenorhabditis elegans ( C. elegans ) strain expressing human WT β 2 -m at high concentrations, mimicking the condition that underlies dialysis-related amyloidosis (DRA) and we compare it to a previously established strain expressing the highly amyloidogenic D76N β 2 -m at lower concentrations. Both strains exhibit behavioral defects, the severity of which correlates with β 2 -m levels rather than with the presence of mutations, being more pronounced in WT β 2 -m worms. β 2 -m expression also has a deep impact on the nematodes' proteomic and metabolic profiles. Most significantly affected processes include protein degradation and stress response, amino acids metabolism, and bioenergetics. Molecular alterations are more pronounced in worms expressing WT β 2 -m at high concentration compared to D76N β 2 -m worms. Altogether, these data show that β 2 -m is a proteotoxic protein in vivo also in its wild-type form, and that concentration plays a key role in modulating pathogenicity. Our transgenic nematodes recapitulate the distinctive features subtending DRA compared to hereditary β 2 -m amyloidosis (high levels of non-mutated β 2 -m vs. normal levels of variant β 2 -m) and provide important clues on the molecular bases of these human diseases., Competing Interests: The authors declare no conflicts of interest., (©2023 The Authors FASEB BioAdvances published by The Federation of American Societies for Experimental Biology.)

Amniotic fluid is essential for fetus wellbeing and is used to monitor pregnancy and predict fetal outcomes. Sex affects health and medicine from the beginning of life, but knowledge of its influence on cell-depleted amniotic fluid (AF) and amniotic fluid cells (AFCs) is still neglected. We evaluated sex-related differences in AF and in AFCs to extend personalized medicine to prenatal life. AFCs and AF were obtained from healthy Caucasian pregnant women who underwent amniocentesis at the 16th-18th week of gestation for advanced maternal age. In the AF, inflammation biomarkers (TNFα, IL6, IL8, and IL4), malondialdehyde, nitrites, amino acids, and acylcarnitines were measured. Estrogen receptors and cell fate (autophagy, apoptosis, senescence) were measured in AFCs. TNFα, IL8, and IL4 were higher in female AF, whereas IL6, nitrites, and MDA were similar. Valine was higher in male AF, whereas several acylcarnitines were sexually different, suggesting a mitochondrial involvement in establishing sex differences. Female AFCs displayed higher expression of ERα protein and a higher ERα/ERβ ratio. The ratio of LC3II/I, an index of autophagy, was higher in female AFCs, while LC3 gene was similar in both sexes. No significant sex differences were found in the expression of the lysosomal protein LAMP1, while p62 was higher in male AFCs. LAMP1 gene was upregulated in male AFCs, while p62 gene was upregulated in female ones. Finally, caspase 9 activity and senescence linked to telomeres were higher in female AFCs, while caspase 3 and β-galactosidase activities were similar. This study supports the idea that sex differences start very early in prenatal life and influence specific parameters, suggesting that it may be relevant to appreciate sex differences to cover knowledge gaps. This might lead to improving the diagnosis of risk prediction for pregnancy complications and achieving a more satisfactory monitoring of fetus health, even preventing future diseases in adulthood.

Introduction: Crohn's disease (CD) is an immune-mediated inflammatory disorder of the gastrointestinal tract with a relapsing-remitting course. Amino acids (AAs) may play critical roles in the intestinal manifestations of disease, due to their involvement in many metabolic and immune functions. The present study aimed to explore serum AA concentrations in adult patients with CD, looking into their variations due to disease activity, surgery and protein content of diet. Eventually, the link between AAs and inflammatory markers was also assessed., Methods: Consecutive adult patients aged 18-65 years with diagnosis of CD were recruited. All participants underwent anthropometry and were instructed to fill in a 3-day food record to assess protein intake. Disease activity was clinically defined using the Crohn's Disease Activity Index (CDAI), while blood samples were taken to analyze serum AA profile and inflammatory markers., Results: A total of 103 patients with CD (61 men and 42 women; age:39.9 ± 13.9 years, BMI: 23.4 ± 3.51 kg/m 2 ) were included. Tryptophan levels were found to be remarkably decreased in most subjects, unrelated to disease activity. On the contrary, concentration of lysine, leucine, valine and glutamine decreased in active versus quiescent CD patients, while aspartic acid, glutamate and glycine increased. The latter AAs were also directly correlated with CDAI and serum interleukin (IL)- 1β concentration. Considering the total protein intake, expressed as g/kg/body weight, we observed a reduction in some essential AAs in patients with unmet protein requirements compared to patients who met the recommendation., Discussion: In conclusion, specific AAs varied according to disease activity and protein intake, adjusted to body weight and disease status. Glu and Asp concentrations raised with increasing IL-1β. However, extensive research is needed to understand the mechanisms underpinning the link between variation in serum AAs, disease activity and protein intake in patients with CD., Competing Interests: MF, FS, and MR were employed by the CEINGE - Biotecnologie Avanzate F. Salvatore, s.c.ar.l. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cioffi, Di Vincenzo, Imperatore, Fisco, Testa, Scialò, Castiglione, Ruoppolo, Pasanisi and Santarpia.)

Background: Glycogen storage disease type Ia (GSDIa) is an inborn metabolic disorder caused by the deficiency of glucose-6-phospatase-α (G6Pase-α) leading to mitochondrial dysfunction. It remains unclear whether mitochondrial dysfunction is present in patients' peripheral blood mononuclear cells (PBMC) and whether dietary treatment can play a role. The aim of this study was to investigate mitochondrial function in PBMC of GSDIa patients., Methods: Ten GSDIa patients and 10 age-, sex- and fasting-time matched controls were enrolled. Expression of genes involved in mitochondrial function and activity of key fatty acid oxidation (FAO) and Krebs cycle proteins were assessed in PBMC. Targeted metabolomics and assessment of metabolic control markers were also performed., Results: Adult GSDIa patients showed increased CPT1A, SDHB, TFAM, mTOR expression (p < 0.05) and increased VLCAD, CPT2 and citrate synthase activity in PBMC (p < 0.05). VLCAD activity directly correlated with WC (p < 0.01), BMI (p < 0.05), serum malonycarnitine levels (p < 0.05). CPT2 activity directly correlated with BMI (p < 0.05)., Conclusion: Mitochondrial reprogramming is detectable in PBMC of GSDIa patients. This feature may develop as an adaptation to the liver enzyme defect and may be triggered by dietary (over)treatment in the frame of G6Pase-α deficiency. PBMC can represent an adequate mean to assess (diet-induced) metabolic disturbances in GSDIa., (© 2023. The Author(s).)

Background: the deficiency of 5,10-Methylenetetrahydrofolate reductase (MTHFR) constitutes a rare and severe metabolic disease and is included in most expanded newborn screening (NBS) programs worldwide. Patients with severe MTHFR deficiency develop neurological disorders and premature vascular disease. Timely diagnosis through NBS allows early treatment, resulting in improved outcomes., Methods: we report the diagnostic yield of genetic testing for MTHFR deficiency diagnosis, in a reference Centre of Southern Italy between 2017 and 2022. MTHFR deficiency was suspected in four newborns showing hypomethioninemia and hyperhomocysteinemia; otherwise, one patient born in pre-screening era showed clinical symptoms and laboratory signs that prompted to perform genetic testing for MTHFR deficiency., Results: molecular analysis of the MTHFR gene revealed a genotype compatible with MTHFR deficiency in two NBS-positive newborns and in the symptomatic patient. This allowed for promptly beginning the adequate metabolic therapy., Conclusions: our results strongly support the need for genetic testing to quickly support the definitive diagnosis of MTHFR deficiency and start therapy. Furthermore, our study extends knowledge of the molecular epidemiology of MTHFR deficiency by identifying a novel mutation in the MTHFR gene.

Gyrate atrophy of choroid and retina (GACR) is a chorioretinal degeneration caused by pathogenic variants in the gene encoding ornithine aminotransferase (OAT), an enzyme mainly expressed in liver. Affected patients have increased ornithine concentrations in blood and other body fluids and develop progressive constriction of vision fields leading to blindness. Current therapies are unsatisfactory and better treatments are highly needed. In two mouse models of OAT deficiency that recapitulates biochemical and retinal changes of GACR, we investigated the efficacy of an intravenously injected serotype 8 adeno-associated (AAV8) vector expressing OAT under the control of a hepatocyte-specific promoter. Following injections, OAT-deficient mice showed reductions of ornithine concentrations in blood and eye cups compared with control mice injected with a vector expressing green fluorescent protein. AAV-injected mice showed improved electroretinogram response and partial restoration of retinal structure up to one-year post-injection. In summary, hepatic OAT expression by AAV8 vector was effective at correction of hyperornithinemia and improved function and structure of the retina. In conclusion, this study provides proof-of-concept of efficacy of liver-directed AAV-mediated gene therapy of GACR., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Cystic fibrosis is a hereditary metabolic disorder characterized by impaired traffic of chloride ions and water through membranes of the respiratory and gastrointestinal, that causes inadequate hydration of airway surfaces, dehydrated mucous secretions and a high-sodium chloride sweat. Although the classical presentation of the condition is well known, a better characterization of metabolic alterations related is need. In particular, the metabolic composition alterations of biological fluids may be influence by the disease state and could be captured as putative signature to set targeted therapeutic strategies. A targeted comprehensive mass spectrometry-based platform was employed to dissect the lipid content of saliva samples form CF patients, in order to investigate alterations in the lipid metabolic homeostasis related to the pathology, chronic obstructive pulmonary disease, Pseudomonas Aeruginosa infection, pancreatic insufficiency, liver disfunction and diabetes-related complications., (© 2023. The Author(s).)

Sexual dimorphism creates different biological and cellular activities and selective regulation mechanisms in males and females, thus generating differential responses in health and disease. In this scenario, the sex itself is a source of physiologic metabolic disparities that depend on constitutive genetic and epigenetic features that characterize in a specific manner one sex or the other. This has as a direct consequence a huge impact on the metabolic routes that drive the phenotype of an individual. The impact of sex is being clearly recognized also in disease, whereas male and females are more prone to the development of some disorders, or have selective responses to drugs and therapeutic treatments. Actually, very less is known regarding the probable differences guided by sex in the context of inherited metabolic disorders, owing to the scarce consideration of sex in such restricted field, accompanied by an intrinsic bias connected with the rarity of such diseases. Metabolomics technologies have been ultimately developed and adopted for being excellent tools for the investigation of metabolic mechanisms, for marker discovery or monitoring, and for supporting diagnostic procedures of metabolic disorders. Hence, metabolomic approaches can excellently embrace the discovery of sex differences, especially when associated to the outcome or the management of certain inborn errors of the metabolism., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Background: Aging and sedentary behavior are independent risk factors for non-communicable diseases. An active lifestyle and structured physical activity are positively associated with a healthier quality of life in the elderly. Here, we explored the proteomic/metabolomic muscular signature induced by lifelong football training associated with successful aging., Methods: The study was performed on nine lifelong football players (67.3 ± 2.8 yrs) and nine aged-matched untrained subjects. We performed a proteomic/metabolomic approach on V. lateralis muscle biopsies; the obtained data were analyzed by means of different bioinformatic tools., Results: Our results indicated that lifelong football training is able to enhance the muscles' oxidative capacity in the elderly by promoting fatty acids as preferential energetic substrates and hence determining a healthier body composition and metabolic profile; furthermore, we showed that the total polyamine content is higher in lifelong football players' muscle, enforcing the involvement of polyamines in muscle growth and hypertrophy., Conclusions: Lifelong football training, as a structured physical activity, significantly influences the expression of the proteins and metabolites involved in oxidative metabolism and muscle hypertrophy associated with successful aging.

Chronic kidney disease (CKD) is a major clinical sign of patients with Bardet-Biedl syndrome (BBS), especially in those carrying BBS10 mutations. Twenty-nine patients with BBS and 30 controls underwent a serum-targeted metabolomic analysis. In vitro studies were conducted in two kidney-derived epithelial cell lines, where Bbs10 was stably deleted (IMCD3- Bbs10 -/-cells) and over-expressed. The CKD status affected plasmatic metabolite fingerprinting in both patients with BBS and controls. Specific phosphatidylcholine and acylcarnitines discriminated eGFR decline only in patients with BBS. IMCD3- Bbs10 -/ cells displayed intracellular lipidaccumulation, reduced mitochondrial potential membrane and citrate synthase staining. Mass-Spectrometry-based analysis revealed that human BBS10 interacted with six mitochondrial proteins, in vitro. In conclusion, renal dysfunction correlated with abnormal phosphatidylcholine and acylcarnitines plasma levels in patients with BBS; in vitro , Bbs10 depletion caused mitochondrial defects while human BBS10 interacted with several mitochondria-related proteins, suggesting an unexplored role of this protein., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Introduction: Short/branched-chain acyl-CoA dehydrogenase deficiency (SBCADD) is an inherited disorder of L-isoleucine metabolism due to mutations in the ACADSB gene. The role of current diagnostic biomarkers [i.e., blood 2-methylbutyrylcarnitine (C5) and urine 2-methylbutyrylglycine (2MBG)] in patient monitoring and the effects of proposed treatments remain uncertain as follow-data are lacking. This study presents first systematic longitudinal biochemical assessment in SBCADD patients., Methods: A retrospective, observational single-center study was conducted on newborns born between 2017 and 2020 and suspected with SBCADD. Biochemical, molecular, clinical and dietary data collected upon NBS recall and during the subsequent follow-up were recorded., Results: All enrolled subjects ( n = 10) received adequate protein intake and L-carnitine supplementation. Nine subjects were diagnosed with SBCADD. During the follow-up [median: 20.5 (4-40) months] no patient developed symptoms related to SBCADD. No patient normalized serum C5 and urine 2MBG values. In 7/9 SBCADD patients mean serum C5 values decreased or stabilized compared to their first serum C5 value. A major increase in serum C5 values was observed in two patients after L-carnitine discontinuation and during intercurrent illness, respectively. Urine 2MBG values showed moderate intra-patient variability., Discussion: The relatively stable serum C5 values observed during L-carnitine supplementation together with C5 increase occurring upon L-carnitine discontinuation/intercurrent illness may support the value of serum C5 as a monitoring biomarker and the benefit of this treatment in SBCADD patients. The role of urine 2MBG in patient monitoring remains uncertain. As all patients were asymptomatic, no association between biochemical parameters and clinical phenotype could be investigated in this study., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rossi, Turturo, Albano, Fecarotta, Barretta, Crisci, Gallo, Perfetto, Uomo, Vallone, Villani, Strisciuglio, Parenti, Frisso and Ruoppolo.)

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive ciliopathy resulting in multiple organ dysfunctions, including chronic kidney disease (CKD). Despite the recent progress in the 'ciliopathy' field, there is still little information on the mechanisms underlying renal disease. To elucidate these pathomechanisms, we conducted a translational study, including (i) the characterization of the urine metabolomic pattern of BBS patients and controls in a pilot and confirmation study and (ii) the proteomic analysis of the BBS10 interactome, one of the major mutated BBS genes in patients, in a renal-epithelial-derived cell culture model. The urine metabolomic fingerprinting of BBS patients differed from controls in both pilot and confirmation studies, demonstrating an increased urinary excretion of several monocarboxylates, including lactic acid (LA), at both early and late CKD stages. Increased urine LA was detected in the absence of both increased plasmatic LA levels and generalized proximal tubular dysfunction, suggesting a possible renal-specific defective handling. The inner medulla renal epithelial (IMCD3) cell line, where Bbs10 was stably invalidated, displayed an increased proliferative rate, increased ATP production, and an up-regulation of aerobic glycolysis. A mass spectrometry-based analysis detected several putative BBS10 interactors in vitro, indicating a potential role of BBS10 in several biological processes, including renal metabolism, RNA processing, and cell proliferation. The present study suggests that the urine metabolomic pattern of BBS patients may reflect intra-renal metabolic aberrations. The analysis of BBS10 interactors unveils possible novel functions, including cell metabolism.

Newborn screening (NBS) for inborn errors of metabolism is one of the most advanced tools for secondary prevention in medicine, as it allows early diagnosis and prompt treatment initiation. The expanded newborn screening was introduced in Italy between 2016 and 2017 (Law 167/2016; DM 13 October 2016; DPCM 12-1-2017). A total of 1,586,578 infants born in Italy were screened between January 2017 and December 2020. For this survey, we collected data from 15 Italian screening laboratories, focusing on the metabolic disorders identified by tandem mass spectrometry (MS/MS) based analysis between January 2019 and December 2020. Aminoacidemias were the most common inborn errors in Italy, and an equal percentage was observed in detecting organic acidemias and mitochondrial fatty acids beta-oxidation defects. Second-tier tests are widely used in most laboratories to reduce false positives. For example, second-tier tests for methylmalonic acid and homocysteine considerably improved the screening of CblC without increasing unnecessary recalls. Finally, the newborn screening allowed us to identify conditions that are mainly secondary to a maternal deficiency. We describe the goals reached since the introduction of the screening in Italy by exchanging knowledge and experiences among the laboratories.

Galactosemia is an inborn disorder of carbohydrate metabolism characterized by the inability to metabolize galactose, a sugar contained in milk (the main source of nourishment for infants), and convert it into glucose, the sugar used by the body as the primary source of energy. Galactosemia is an autosomal recessive genetic disease that can be diagnosed at birth, even in the absence of symptoms, with newborn screening by assessing the level of galactose and the GALT enzyme activity, as GALT defect constitutes the most frequent cause of galactosemia. Currently, galactosemia cannot be cured, but only treated by means of a diet with a reduced content of galactose and lactose. Although the diet is able to reverse the neonatal clinical picture, it does not prevent the development of long-term complications. This review provides an overview of galactose metabolism, molecular genetics, newborn screening and therapy of galactosemia. Novel treatments for galactosemia currently being investigated in (pre)clinical studies and potentially able to prevent long-term complications are also presented.

Background: The sexual dimorphism represents one of the triggers of the metabolic disparities between the organisms, advising about wild implications in research or diagnostics contexts. Despite the mounting recognition of the importance of sex consideration in the biomedical fields, the identification of male- and female-specific metabolic signatures has not been achieved., Main Body: This review pointed the focus on the metabolic differences related to the sex, evidenced by metabolomics studies performed on healthy populations, with the leading aim of understanding how the sex influences the baseline metabolome. The main shared signatures and the apparent dissimilarities between males and females were extracted and highlighted from the metabolome of the most commonly analyzed biological fluids, such as serum, plasma, and urine. Furthermore, the influence of age and the significant interactions between sex and age have been taken into account., Conclusions: The recognition of sex patterns in human metabolomics has been defined in diverse biofluids. The detection of sex- and age-related differences in the metabolome of healthy individuals are helpful for translational applications from the bench to the bedside to set targeted diagnostic and prevention approaches in the context of personalized medicine., (© 2022. The Author(s).)

X-linked lissencephaly with abnormal genitalia (XLAG) and developmental epileptic encephalopathy-1 (DEE1) are caused by mutations in the Aristaless-related homeobox (ARX) gene, which encodes a transcription factor responsible for brain development. It has been unknown whether the phenotypically diverse XLAG and DEE1 phenotypes may converge on shared pathways. To address this question, a label-free quantitative proteomic approach was applied to the neonatal brain of Arx knockout (ArxKO/Y) and knock-in polyalanine (Arx(GCG)7/Y) mice that are respectively models for XLAG and DEE1. Gene ontology and protein-protein interaction analysis revealed that cytoskeleton, protein synthesis and splicing control are deregulated in an allelic-dependent manner. Decreased α-tubulin content was observed both in Arx mice and Arx/alr-1(KO) Caenorhabditis elegans ,and a disorganized neurite network in murine primary neurons was consistent with an allelic-dependent secondary tubulinopathy. As distinct features of Arx(GCG)7/Y mice, we detected eIF4A2 overexpression and translational suppression in cortex and primary neurons. Allelic-dependent differences were also established in alternative splicing (AS) regulated by PUF60 and SAM68. Abnormal AS repertoires in Neurexin-1, a gene encoding multiple pre-synaptic organizers implicated in synaptic remodelling, were detected in Arx/alr-1(KO) animals and in Arx(GCG)7/Y epileptogenic brain areas and depolarized cortical neurons. Consistent with a conserved role of ARX in modulating AS, we propose that the allelic-dependent secondary synaptopathy results from an aberrant Neurexin-1 repertoire. Overall, our data reveal alterations mirroring the overlapping and variant effects caused by null and polyalanine expanded mutations in ARX. The identification of these effects can aid in the design of pathway-guided therapy for ARX endophenotypes and NDDs with overlapping comorbidities., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Metabolomics helps identify metabolites to characterize/refine perturbations of biological pathways in living organisms. Pre-analytical, analytical, and post-analytical limitations that have hampered a wide implementation of metabolomics have been addressed. Several potential biomarkers originating from current targeted metabolomics-based approaches have been discovered. Precision medicine argues for algorithms to classify individuals based on susceptibility to disease, and/or by response to specific treatments. It also argues for a prevention-based health system. Because of its ability to explore gene-environment interactions, metabolomics is expected to be critical to personalize diagnosis and treatment. Stringent guidelines have been applied from the very beginning to design studies to acquire the information currently employed in precision medicine and precision prevention approaches. Large, prospective, expensive and time-consuming studies are now mandatory to validate old, and discover new, metabolomics-based biomarkers with high chances of translation into precision medicine. Metabolites from studies on saliva, sweat, breath, semen, feces, amniotic, cerebrospinal, and broncho-alveolar fluid are predicted to be needed to refine information from plasma and serum metabolome. In addition, a multi-omics data analysis system is predicted to be needed for omics -based precision medicine approaches. Omics -based approaches for the progress of precision medicine and prevention are expected to raise ethical issues.

Omics-based technologies have been largely adopted during this unprecedented global COVID-19 pandemic, allowing the scientific community to perform research on a large scale to understand the pathobiology of the SARS-CoV-2 infection and its replication into human cells. The application of omics techniques has been addressed to every level of application, from the detection of mutations, methods of diagnosis or monitoring, drug target discovery, and vaccine generation, to the basic definition of the pathophysiological processes and the biochemical mechanisms behind the infection and spread of SARS-CoV-2. Thus, the term COVIDomics wants to include those efforts provided by omics-scale investigations with application to the current COVID-19 research. This review summarizes the diverse pieces of knowledge acquired with the application of COVIDomics techniques, with the main focus on proteomics and metabolomics studies, in order to capture a common signature in terms of proteins, metabolites, and pathways dysregulated in COVID-19 disease. Exploring the multiomics perspective and the concurrent data integration may provide new suitable therapeutic solutions to combat the COVID-19 pandemic.

In this article, we present data on the proteome of human neuroblastoma cells stably overexpressing Neuroglobin (NGB). The neuroprotective role of NGB is clearly established, nevertheless the related mechanistic processes, which are dependent on NGB overexpression, are not known. To address this question, we performed shotgun label-free quantification (LFQ) proteomics using an SH-SY5Y cell model of neuroblastoma that overexpresses an NGB-FLAG construct, and wild type cells transfected with an empty vector as control (CTRL). The proteomes from six biological samples per condition were digested using the S-Trap sample preparation followed by LC-MS/MS analysis with a LTQ-Orbitrap XL mass spectrometer. The quantitative analysis was performed using the LFQ algorithm of MaxQuant, leading to 1654 correctly quantified proteins over 2580 identified proteins. Finally, the statistic comparison of the two analyzed groups within Perseus platform identified 178 differential proteins (107 up- and 71 down-regulated). In addition, multivariate statistical analysis was carried out using MetaboAnalyst 5.0 software. MS proteomics data are available via ProteomeXchange with the dataset identifier PXD029012., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s). Published by Elsevier Inc.)

Neuroglobin (NGB) is an O 2 -binding globin mainly expressed in the central and peripheral nervous systems and cerebrospinal fluid. Previously, it was demonstrated that NGB overexpression protects cells from hypoxia-induced death. To investigate processes promoted by NGB overexpression, we used a cellular model of neuroblastoma stably overexpressing an NGB-FLAG construct. We used a proteomic approach to identify the specific profile following NGB overexpression. To evaluate the role of NGB overexpression in increasing energetic metabolism, we measured oxygen consumption rate (OCR) and the extracellular acidification rate through Seahorse XF technology. The effect on autophagy induction was evaluated by analyzing SQSTM1/p62 and LC3-II expression. Proteomic analysis revealed several differentially regulated proteins, involved in oxidative phosphorylation and integral mitochondrial proteins linked to energy metabolism. The analysis of mitochondrial metabolism demonstrated that NGB overexpression increases mitochondrial ATP production. Indeed, NGB overexpression enhances bioenergetic metabolism, increasing OCR and oxygen consumption. Analysis of autophagy induction revealed an increase of LC3-II together with a significant decrease of SQSTM1/p62, and NGB-LC3-II association during autophagosome formation. These results highlight the active participation of NGB in several cellular processes that can be upregulated in response to NGB overexpression, playing a role in the adaptive response to stress in neuroblastoma cells.

Glutaric aciduria type I (GA-I, OMIM # 231670) is an autosomal recessive inborn error of metabolism caused by deficiency of the mitochondrial enzyme glutaryl-CoA dehydrogenase (GCDH). The principal clinical manifestation in GA-I patients is striatal injury most often triggered by catabolic stress. Early diagnosis by newborn screening programs improved survival and reduced striatal damage in GA-I patients. However, the clinical phenotype is still evolving in the aging patient population. Evaluation of long-term outcome in GA-I patients recently identified glomerular filtration rate (GFR) decline with increasing age. We recently created the first knock-in rat model for GA-I harboring the mutation p.R411W (c.1231 C>T), corresponding to the most frequent GCDH human mutation p.R402W. In this study, we evaluated the effect of an acute metabolic stress in form of high lysine diet (HLD) on young Gcdh ki/ki rats. We further studied the chronic effect of GCDH deficiency on kidney function in a longitudinal study on a cohort of Gcdh ki/ki rats by repetitive 68 Ga-EDTA positron emission tomography (PET) renography, biochemical and histological analyses. In young Gcdh ki/ki rats exposed to HLD, we observed a GFR decline and biochemical signs of a tubulopathy. Histological analyses revealed lipophilic vacuoles, thinning of apical brush border membranes and increased numbers of mitochondria in proximal tubular (PT) cells. HLD also altered OXPHOS activities and proteome in kidneys of Gcdh ki/ki rats. In the longitudinal cohort, we showed a progressive GFR decline in Gcdh ki/ki rats starting at young adult age and a decline of renal clearance. Histopathological analyses in aged Gcdh ki/ki rats revealed tubular dilatation, protein accumulation in PT cells and mononuclear infiltrations. These observations confirm that GA-I leads to acute and chronic renal damage. This raises questions on indication for follow-up on kidney function in GA-I patients and possible therapeutic interventions to avoid renal damage., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The mainstay of phenylketonuria treatment is a low protein diet, supplemented with phenylalanine (Phe)-free protein substitutes and micronutrients. Adhering to this diet is challenging, and even patients with good metabolic control who follow the dietary prescriptions in everyday life ignore the recommendations occasionally. The present study explores the ability of slow-release large neutral amino acids (srLNAAs) to prevent Phe increase following a Phe dietary load. Fourteen phenylketonuric patients aged ≥13 years were enrolled in a 6-week protocol. Oral acute Phe loads of 250 and 500 mg were added to the evening meal together with srLNAAs (0.5 gr/kg). Phe and tyrosine were dosed before dinner, 2h-after dinner, and after the overnight fast. After oral Phe loads, mean plasma Phe remained stable and below 600 µmol/L. No Phe peaks were registered. Tyrosine levels significantly increased, and Phe/Tyrosine ratio decreased. No adverse events were registered. In conclusion, a single oral administration of srLNAAs at the dose of 0.5 gr/kg is effective in maintaining stable plasma Phe during acute oral loads with Phe-containing food and may be added to the dietetic scheme in situations in which patients with generally good adherence to diet foresee a higher than prescribed Phe intake due to their commitments.

COVID-19 is a global threat that has spread since the end of 2019, causing severe clinical sequelae and deaths, in the context of a world pandemic. The infection of the highly pathogenetic and infectious SARS-CoV-2 coronavirus has been proven to exert systemic effects impacting the metabolism. Yet, the metabolic pathways involved in the pathophysiology and progression of COVID-19 are still unclear. Here, we present the results of a mass spectrometry-based targeted metabolomic analysis on a cohort of 52 hospitalized COVID-19 patients, classified according to disease severity as mild, moderate, and severe. Our analysis defines a clear signature of COVID-19 that includes increased serum levels of lactic acid in all the forms of the disease. Pathway analysis revealed dysregulation of energy production and amino acid metabolism. Globally, the variations found in the serum metabolome of COVID-19 patients may reflect a more complex systemic perturbation induced by SARS-CoV-2, possibly affecting carbon and nitrogen liver metabolism.

Aims: Untargeted Metabolomics is a "hypothesis-generating discovery strategy" that compares groups of samples (e.g., cases vs controls); identifies the metabolome and establishes (early signs of) perturbations. Targeted Metabolomics helped gather key information in life sciences and disclosed novel strategies for the treatment of major clinical entities (e.g., malignancy, cardiovascular diabetes mellitus, drug toxicity). Because of its relevance in biomarker discovery, attention is now devoted to improving the translational potential of untargeted Metabolomics., Data Synthesis: Expertise in laboratory medicine and in bioinformatics helps solve challenges/pitfalls that may bias metabolite profiling in untargeted Metabolomics. Clinical validation (availability/reliability of analytical instruments) and profitability (how many people will use the test) are mandatory steps for potential biomarkers. Biomarkers to predict individual patient response, patient populations that will best respond to specific strategies and/or approaches for an optimal response to treatment are now being developed. Additional help is expected from professional, and regulatory Agencies as to guidelines for study design and data acquisition and analysis, to be applied from the very beginning of a project. Evidence from food, plant, human, environmental, and animal research argues for the need of miniaturized approaches that employ low-cost, easy to use, mobile devices. ELISA kits with such characteristics that employ targeted metabolites are already available., Conclusions: Improving knowledge of the mechanisms behind the disease status (pathophysiology) will help untargeted Metabolomics gather a direct positive impact on welfare and industrial advancements, and fade uncertainties perceived by regulators/payers and patients concerning variables related to miniaturised instruments and user-friendly software and databases., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2021. Published by Elsevier B.V.)

Glutaric aciduria type I (GA-I, OMIM # 231670) is an inborn error of metabolism caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients develop acute encephalopathic crises (AEC) with striatal injury most often triggered by catabolic stress. The pathophysiology of GA-I, particularly in brain, is still not fully understood. We generated the first knock-in rat model for GA-I by introduction of the mutation p.R411W, the rat sequence homologue of the most common Caucasian mutation p.R402W, into the Gcdh gene of Sprague Dawley rats by CRISPR/CAS9 technology. Homozygous Gcdh ki/ki rats revealed a high excretor phenotype, but did not present any signs of AEC under normal diet (ND). Exposure to a high lysine diet (HLD, 4.7%) after weaning resulted in clinical and biochemical signs of AEC. A significant increase of plasmatic ammonium concentrations was found in Gcdh ki/ki rats under HLD, accompanied by a decrease of urea concentrations and a concomitant increase of arginine excretion. This might indicate an inhibition of the urea cycle. Gcdh ki/ki rats exposed to HLD showed highly diminished food intake resulting in severely decreased weight gain and moderate reduction of body mass index (BMI). This constellation suggests a loss of appetite. Under HLD, pipecolic acid increased significantly in cerebral and extra-cerebral liquids and tissues of Gcdh ki/ki rats, but not in WT rats. It seems that Gcdh ki/ki rats under HLD activate the pipecolate pathway for lysine degradation. Gcdh ki/ki rat brains revealed depletion of free carnitine, microglial activation, astroglyosis, astrocytic death by apoptosis, increased vacuole numbers, impaired OXPHOS activities and neuronal damage. Under HLD, Gcdh ki/ki rats showed imbalance of intra- and extracellular creatine concentrations and indirect signs of an intracerebral ammonium accumulation. We successfully created the first rat model for GA-I. Characterization of this Gcdh ki/ki strain confirmed that it is a suitable model not only for the study of pathophysiological processes, but also for the development of new therapeutic interventions. We further brought up interesting new insights into the pathophysiology of GA-I in brain and periphery., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Neuroglobin (NGB) is a myoglobin-like monomeric globin that is involved in several processes, displaying a pivotal redox-dependent protective role in neuronal and extra-neuronal cells. NGB remarkably exerts its function upon upregulation by NGB inducers, such as 17β-estradiol (E2) and H 2 O 2 . However, the molecular bases of NGB's functions remain undefined, mainly in non-neuronal cancer cells. Human MCF-7 breast cancer cells with a knocked-out (KO) NGB gene obtained using CRISPR/Cas9 technology were analyzed using shotgun label-free quantitative proteomics in comparison with control cells. The differential proteomics experiments were also performed after treatment with E2, H 2 O 2 , and E2 + H 2 O 2 . All the runs acquired using liquid chromatography-tandem mass spectrometry were elaborated within the same MaxQuant analysis, leading to the quantification of 1872 proteins in the global proteomic dataset. Then, a differentially regulated protein dataset was obtained for each specific treatment. After the proteomic study, multiple bioinformatics analyses were performed to highlight unbalanced pathways and processes. Here, we report the proteomic and bioinformatic investigations concerning the effects on cellular processes of NGB deficiency and cell treatments. Globally, the main processes that were affected were related to the response to stress, cytoskeleton dynamics, apoptosis, and mitochondria-driven pathways.

Prematurity is the leading cause of neonatal deaths and high economic costs; it depends on numerous biological and social factors, and is highly prevalent in males. Several factors can affect the metabolome of premature infants. Accordingly, the aim of the present study was to analyze the role played by gestational age (GA), parenteral nutrition (PN), and caffeine treatment in sex-related differences of blood metabolome of premature neonates through a MS/MS-based targeted metabolomic approach for the detection of amino acids and acylcarnitines in dried blood spots. GA affected the blood metabolome of premature neonates: male and female very premature infants (VPI) diverged in amino acids but not in acylcarnitines, whereas the opposite was observed in moderate or late preterm infants (MLPI). Moreover, an important reduction of metabolites was observed in female VPI fed with PN, suggesting that PN might not satisfy an infant's nutritional needs. Caffeine showed the highest significant impact on metabolite levels of male MLPI. This study proves the presence of a sex-dependent metabolome in premature infants, which is affected by GA and pharmacological treatment (e.g., caffeine). Furthermore, it describes an integrated relationship among several features of physiology and health.

In recent months, Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread throughout the world. COVID-19 patients show mild, moderate or severe symptoms with the latter ones requiring access to specialized intensive care. SARS-CoV-2 infections, pathogenesis and progression have not been clearly elucidated yet, thus forcing the development of many complementary approaches to identify candidate cellular pathways involved in disease progression. Host lipids play a critical role in the virus life, being the double-membrane vesicles a key factor in coronavirus replication. Moreover, lipid biogenesis pathways affect receptor-mediated virus entry at the endosomal cell surface and modulate virus propagation. In this study, targeted lipidomic analysis coupled with proinflammatory cytokines and alarmins measurement were carried out in serum of COVID-19 patients characterized by different severity degree. Serum IL-26, a cytokine involved in IL-17 pathway, TSLP and adiponectin were measured and correlated to lipid COVID-19 patient profiles. These results could be important for the classification of the COVID-19 disease and the identification of therapeutic targets.

The primary cilium (PC) was considered as a vestigial organelle with no significant physiological importance, until the discovery that PC perturbation disturbs several signalling pathways and results in the dysfunction of a variety of organs. Genetic studies have demonstrated that mutations affecting PC proteins or its anchoring structure, the basal body, underlie a class of human disorders (known as ciliopathies) characterized by a constellation of clinical signs. Further investigations have demonstrated that the PC is involved in a broad range of biological processes, in both developing and mature tissues. Kidney disease is a common clinical feature of cilia disorders, supporting the hypothesis of a crucial role of the PC in kidney homoeostasis. Clinical proteomics and metabolomics are an expanding research area. Interestingly, the application of these methodologies to the analysis of urine, a biological sample that can be collected in a non-invasive fashion and possibly in large amounts, makes these studies feasible also in patients. The present article describes the most recent proteomic and metabolomic studies exploring kidney dysfunction in the setting of ciliopathies, showing the potential of these methodologies in the elucidation of disease pathophysiology and in the discovery of biomarkers., (© The Author(s) 2019. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Methylmalonic acidemia is a rare inborn error of metabolism with severe clinical complications and poor outcome. The present data article is related to a proteomic investigation conducted on a HEK 293 cell line which has been genetically modified using CRISPR-CAS9 system to knockout the methylmalonyl-CoA mutase enzyme (MUT-KO). Thus, the generated cell model for methylmalonic acidemia was used for a proteomic comparison with respect to HEK 293 wild type cells performing a label-free quantification (LFQ) experiment. A comparison between FASP and S-Trap digestion methods was performed on protein extracts before to proceed with the proteomic analysis of the samples. Four biological replicates were employed for LC-MS/MS analysis and each was run in technical triplicates. MaxQuant and Perseus platforms were used to perform the LFQ of the proteomes and carry out statistical analysis, respectively. Globally, 4341 proteins were identified, and 243 as differentially regulated, of which 150 down-regulated and 93 up-regulated in the MUT-KO condition. MS proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD017977. The information provided in this dataset shed new light on the cellular mechanisms altered in this rare metabolic disorder, highlighting quantitative unbalances in proteins acting in cell structure and architecture organization and response to the stress. This article can be used as a new source of protein actors to be validated and a starting point for the identification of clinically relevant therapeutic targets., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article., (© 2020 The Authors. Published by Elsevier Inc.)

Methylmalonic acidemia (MMA) is a rare inborn error of metabolism caused by deficiency of the methylmalonyl-CoA mutase (MUT) enzyme. Downstream MUT deficiency, methylmalonic acid accumulates together with toxic metabolites from propionyl-CoA and other compounds upstream of the block in the enzyme pathway. The presentation is with life-threatening acidosis, respiratory distress, brain disturbance, hyperammonemia, and ketosis. Survivors develop poorly understood multi-organ damage, notably to the brain and kidneys. The HEK 293 cell line was engineered by CRISPR/Cas9 technology to knock out the MUT gene (MUT-KO). Shotgun label-free quantitative proteomics and bioinformatics analyses revealed potential damaging biological processes in MUT-deficient cells. MUT-KO induced alteration of cellular architecture and morphology, and ROS overproduction. We found the alteration of proteins involved in cytoskeleton and cell adhesion organization, cell trafficking, mitochondrial, and oxidative processes, as validated by the regulation of VIM, EXT2, SDC2, FN1, GLUL, and CHD1. Additionally, a cell model of MUT-rescuing was developed in order to control the specificity of MUT-KO effects. Globally, the proteomic landscape of MUT-KO suggests the cell model to have an increased susceptibility to propionate- and H 2 O 2 -induced stress through an impairment of the mitochondrial functionality and unbalances in the oxidation-reduction processes.

Bardet-Biedl syndrome (BBS) is a rare pleiotropic inherited disorder known as a ciliopathy. Kidney disease is a cardinal clinical feature; however, it is one of the less investigated traits. This study is a comprehensive analysis of the literature aiming to collect available information providing mechanistic insights into the pathogenesis of kidney disease by analyzing clinical and basic science studies focused on this issue. The analysis revealed that the syndrome is either clinically and genetically heterogenous, with 24 genes discovered to date, but with 3 genes ( BBS1 , BBS2 , and BBS10 ) accounting for almost 50% of diagnoses; genotype-phenotype correlation studies showed that patients with BBS1 mutations have a less severe renal phenotype than the other 2 most common loci; in addition, truncating rather than missense mutations are more likely to cause kidney disease. However, significant intrafamilial clinical variability has been described, with no clear explanation to date. In mice kidneys, Bbs genes have relative low expression levels, in contrast with other common affected organs, like the retina; surprisingly, Bbs1 is the only locus with basal overexpression in the kidney. In vitro studies indicate that signalling pathways involved in embryonic kidney development and repair are affected in the context of BBS depletion; in mice, kidney disease does not have a full penetrance; when present, it resembles human phenotype and shows an age-dependent progression. Data on the exact contribution of local versus systemic consequences of Bbs dysfunction are scanty and further investigations are required to get firm conclusions., (© 2020 International Society of Nephrology. Published by Elsevier Inc.)

Mucopolysaccharidoses (MPSs) are inherited disorders of the glycosaminoglycan (GAG) metabolism. The defective digestion of GAGs within the intralysosomal compartment of affected patients leads to a broad spectrum of clinical manifestations ranging from cardiovascular disease to neurological impairment. The molecular mechanisms underlying the progression of the disease downstream of the genetic mutation of genes encoding for lysosomal enzymes still remain unclear. Here, we applied a targeted metabolomic approach to a mouse model of PS IIIB, using a platform dedicated to the diagnosis of inherited metabolic disorders, in order to identify amino acid and fatty acid metabolic pathway alterations or the manifestations of other metabolic phenotypes. Our analysis highlighted an increase in the levels of branched-chain amino acids (BCAAs: Val, Ile, and Leu), aromatic amino acids (Tyr and Phe), free carnitine, and acylcarnitines in the liver and heart tissues of MPS IIIB mice as compared to the wild type (WT). Moreover, Ala, Met, Glu, Gly, Arg, Orn, and Cit amino acids were also found upregulated in the liver of MPS IIIB mice. These findings show a specific impairment of the BCAA and fatty acid catabolism in the heart of MPS IIIB mice. In the liver of affected mice, the glucose-alanine cycle and urea cycle resulted in being altered alongside a deregulation of the BCAA metabolism. Thus, our data demonstrate that an accumulation of BCAAs occurs secondary to lysosomal GAG storage, in both the liver and the heart of MPS IIIB mice. Since BCAAs regulate the biogenesis of lysosomes and autophagy mechanisms through mTOR signaling, impacting on lipid metabolism, this condition might contribute to the progression of the MPS IIIB disease.

Cultured mouse embryonic stem cells are a heterogeneous population with diverse differentiation potential. In particular, the subpopulation marked by Zscan4 expression has high stem cell potency and shares with 2 cell stage preimplantation embryos both genetic and epigenetic mechanisms that orchestrate zygotic genome activation. Although embryonic de novo genome activation is known to rely on metabolites, a more extensive metabolic characterization is missing. Here we analyze the Zscan4 + mouse stem cell metabolic phenotype associated with pluripotency maintenance and cell reprogramming. We show that Zscan4 + cells have an oxidative and adaptable metabolism, which, on one hand, fuels a high bioenergetic demand and, on the other hand, provides intermediate metabolites for epigenetic reprogramming. Our findings enhance our understanding of the metastable Zscan4 + stem cell state with potential applications in regenerative medicine., (© 2020 The Authors.)

Mucopolysaccharidosis IIIB (MPS IIIB) is an inherited metabolic disease due to deficiency of α-N-Acetylglucosaminidase (NAGLU) enzyme with subsequent storage of undegraded heparan sulfate (HS). The main clinical manifestations of the disease are profound intellectual disability and neurodegeneration. A label-free quantitative proteomic approach was applied to compare the proteome profile of brains from MPS IIIB and control mice to identify altered neuropathological pathways of MPS IIIB. Proteins were identified through a bottom up analysis and 130 were significantly under-represented and 74 over-represented in MPS IIIB mouse brains compared to wild type (WT). Multiple bioinformatic analyses allowed to identify three major clusters of the differentially abundant proteins: proteins involved in cytoskeletal regulation, synaptic vesicle trafficking, and energy metabolism. The proteome profile of NAGLU -/- mouse brain could pave the way for further studies aimed at identifying novel therapeutic targets for the MPS IIIB. Data are available via ProteomeXchange with the identifier PXD017363.

Exercise under fasting conditions induces a switch to lipid metabolism, eliciting beneficial metabolic effects. Knowledge of signaling responses underlying metabolic adjustments in such conditions may help to identify therapeutic strategies. Therefore, we studied the effect of mild exercise on rats submitted to food withdrawal at thermoneutrality (28°C) for 3 days. Animals were housed at thermoneutrality rather than the standard housing temperature (22°C) to avoid beta-adrenergic signaling responses that themselves affect metabolism and well-being. Quantitative analysis of multi-organ mRNA levels, myofibers, and serum metabolites shows that this protocol (a) boosts fat oxidation in muscle and liver, (b) reduces lipogenesis and increases gluconeogenesis in liver, (c) increases serum acylcarnitines (especially C 4 OH) and ketone bodies and the use of the latter as fuel in muscle, (d) increases Type I myofibers, and (e) is associated with an increased thyroid hormone uptake and metabolism in muscle. In addition, stool microbiome DNA analysis revealed that food withdrawal dramatically alters the presence of bacterial genera associated with ketone metabolism. Taken together, this protocol induces a drastic switch toward increased lipid and ketone metabolism compared to exercise or food withdrawal alone, which may prove beneficial and may involve local thyroid hormones, which may be regarded as exercise mimetics., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

The characterization of urinary metabolome, which provides a fingerprint for each individual, is an important step to reach personalized medicine. It is influenced by exogenous and endogenous factors; among them, we investigated sex influences on 72 organic acids measured through GC-MS analysis in the urine of 291 children (152 males; 139 females) aging 1-36 months and stratified in four groups of age. Among the 72 urinary metabolites, in all age groups, 4-hydroxy-butirate and homogentisate are found only in males, whereas 3-hydroxy-dodecanoate, methylcitrate, and phenylacetate are found only in females. Sex differences are still present after age stratification being more numerous during the first 6 months of life. The most relevant sex differences involve the mitochondria homeostasis. In females, citrate cycle, glyoxylate and dicarboxylate metabolism, alanine, aspartate, glutamate, and butanoate metabolism had the highest impact. In males, urinary organic acids were involved in phenylalanine metabolism, citrate cycle, alanine, aspartate and glutamate metabolism, butanoate metabolism, and glyoxylate and dicarboxylate metabolism. In addition, age specifically affected metabolic pathways, the phenylalanine metabolism pathway being affected by age only in males. Relevantly, the age-influenced ranking of metabolic pathways varied in the two sexes. In conclusion, sex deeply influences both quantitatively and qualitatively urinary organic acids levels, the effect of sex being age dependent. Importantly, the sex effects depend on the single organic acid; thus, in some cases the urinary organic acid reference values should be stratified according the sex and age., Competing Interests: The authors declare no conflict of interest.

Using differentiated rat L6 cells, we studied the direct effect of 3,5,3'-triiodo-l-thyronine (T3) and 3,5-diiodo-l-thyronine (T2) on the response to insulin in presence of fatty acids with a varying degree of saturation. We found that T3 and T2 both invert the response to insulin by modulating Akt Ser473 phosphorylation in the presence of palmitate and oleate. Both hormones prevented palmitate-induced insulin resistance, whereas increased insulin sensitivity in the presence of oleate was reduced, with normalization to (or, in the case of T3, even below) control levels. Both hormones effectively reduced intracellular acylcarnitine concentrations. Interestingly, insulin sensitization was lowered by incubation of the myotubes with relevant concentrations of palmitoylcarnitines (C16) and increased by oleylcarnitines and linoleylcarnitines (C18:1 and C18:2, respectively). The efficiency of mitochondrial respiration decreased in the order palmitate-oleate-linoleate; in the presence of palmitate, only T3 increased ATP synthesis-independent cellular respiration and mitochondrial respiratory complex activities. Both hormones modulated gene expression and enzyme activities related to insulin sensitivity, glucose metabolism, and lipid handling. Although T2 and T3 differentially regulated the expression of relevant genes involved in glucose metabolism, they equally stimulated related metabolic activities. T2 and T3 differentially modulated mitochondrial fatty acid uptake and oxidation in the presence of each fatty acid. The results show that T2 and T3 both invert the fatty acid-induced response to insulin but through different mechanisms, and that the outcome depends on the degree of saturation of the fatty acids and their derived acylcarnitines.-Giacco, A., delli Paoli, G., Senese, R., Cioffi, F., Silvestri, E., Moreno, M., Ruoppolo, M., Caterino, M., Costanzo, M., Lombardi, A., Goglia, F., Lanni, A., de Lange, P. The saturation degree of fatty acids and their derived acylcarnitines determines the direct effect of metabolically active thyroid hormones on insulin sensitivity in skeletal muscle cells.

Methylmalonic acidemias (MMAs) are inborn errors of metabolism due to the deficient activity of methylmalonyl-CoA mutase (MUT). MUT catalyzes the formation of succinyl-CoA from methylmalonyl-CoA, produced from propionyl-CoA catabolism and derived from odd chain fatty acids β-oxidation, cholesterol, and branched-chain amino acids degradation. Increased methylmalonyl-CoA levels allow for the presymptomatic diagnosis of the disease, even though no approved therapies exist. MMA patients show hyperammonemia, ketoacidosis, lethargy, respiratory distress, cognitive impairment, and hepatomegaly. The long-term consequences concern neurologic damage and terminal kidney failure, with little chance of survival. The cellular pathways affected by MUT deficiency were investigated using a quantitative proteomics approach on a cellular model of MUT knockdown. Currently, a consistent reduction of the MUT protein expression was obtained in the neuroblastoma cell line (SH-SY5Y) by using small-interfering RNA (siRNA) directed against an MUT transcript (MUT siRNA). The MUT absence did not affect the cell viability and apoptotic process in SH-SY5Y. In the present study, we evaluate and quantify the alterations in the protein expression profile as a consequence of MUT-silencing by a mass spectrometry-based label-free quantitative analysis, using two different quantitative strategies. Both quantitative methods allowed us to observe that the expression of the proteins involved in mitochondrial oxido-reductive homeostasis balance was affected by MUT deficiency. The alterated functional mitochondrial activity was observed in siRNA&#95;MUT cells cultured with a propionate-supplemented medium. Finally, alterations in the levels of proteins involved in the metabolic pathways, like carbohydrate metabolism and lipid metabolism, were found.