Search

Highlights • Analysis of 2-methylcitric acid, methylmalonic acid, and homocysteine in DBS. • A dual derivatization LC-MS/MS strategy developed for use in second-tier NBS. • Second-tier panel replaces multiple initial follow-up tests. • Improved workflows without the use of ion pairing reagents in the LC mobile phases. • Anticipated increase in diagnostic specificity and reduction of parental anxiety., Inborn errors of propionate, cobalamin and methionine metabolism are targets for Newborn Screening (NBS) in most programs world-wide, and are primarily screened by analyzing for propionyl carnitine (C3) and methionine in dried blood spot (DBS) cards using tandem mass spectrometry (MS/MS). Single-tier NBS approaches using C3 and methionine alone lack specificity, which can lead to an increased false-positive rate if conservative cut-offs are applied to minimize the risk of missing cases. Implementation of liquid chromatography tandem mass spectrometry (LC-MS/MS) second-tier testing for 2-methylcitric acid (MCA), methylmalonic acid (MMA), and homocysteine (HCY) from the same DBS card can improve disease screening performance by reducing the false-positive rate and eliminating the need for repeat specimen collection. However, DBS analysis of MCA, MMA, and HCY by LC-MS/MS is challenging due to limited specimen size and analyte characteristics leading to a combination of low MS/MS sensitivity and poor reverse-phase chromatographic retention. Sufficient MS response and analytical performance can be achieved for MCA by amidation using DAABD-AE and by butylation for MMA and HCY. Herein we describe the validation of a second-tier dual derivatization LC-MS/MS approach to detect elevated MCA, MMA, and HCY in DBS cards for NBS. Clinical utility was demonstrated by retrospective analysis of specimens, an interlaboratory method comparison, and assessment of external proficiency samples. Imprecision was

Graphical abstract, Introduction Hydrogen sulphide (H2S) has been established as a key member of the gasotransmitters family that recently showed a pivotal role in various pathological conditions including cancer. Objectives This study investigated the role of H2S in breast cancer (BC) pathogenesis, on BC immune recognition capacity and the consequence of targeting H2S using non-coding RNAs. Methods Eighty BC patients have been recruited for the study. BC cell lines were cultured and transfected using validated oligonucleotide delivery system. Gene and protein expression analysis was performed using qRT-PCR, western blot and flow-cytometry. In-vitro analysis for BC hallmarks was performed using MTT, BrdU, Modified Boyden chamber, migration and colony forming assays. H2S and nitric oxide (NO) levels were measured spectrophotometrically. Primary natural killer cells (NK cells) and T cell isolation and chimeric antigen receptor transduction (CAR T cells) were performed using appropriate kits. NK and T cells cytotoxicity was measured. Finally, computational target prediction analysis and binding confirmation analyses were performed using different software and dual luciferase assay kit, respectively. Results The H2S synthesizing enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), exhibited elevated levels in the clinical samples that correlated with tumor proliferation index. Knock-down of CBS and CSE in the HER2+ BC and triple negative BC (TNBC) cells resulted in significant attenuation of BC malignancy. In addition to increased susceptibility of HER2+ BC and TNBC to the cytotoxic activity of HER2 targeting CAR T cells and NK cells, respectively. Transcriptomic and phosphoprotein analysis revealed that H2S signaling is mediated through Akt in MCF7, STAT3 in MDA-MB-231 and miR-155/ NOS2/NO signaling in both cell lines. Lastly, miR-4317 was found to function as an upstream regulator of CBS and CSE synergistically abrogates the malignancy of BC cells. Conclusion These findings demonstrate the potential role of H2S signaling in BC pathogenesis and the potential of its targeting for disease mitigation.

Summary Gene therapy is becoming an increasingly valuable tool to treat many genetic diseases with no or limited treatment options. This is the case for hundreds of monogenic metabolic disorders of hepatic origin, for which liver transplantation remains the only cure. Furthermore, the liver contains 10–15% of the body’s total blood volume, making it ideal for use as a factory to secrete proteins into the circulation. In recent decades, an expanding toolbox has become available for liver-directed gene delivery. Although viral vectors have long been the preferred approach to target hepatocytes, an increasing number of non-viral vectors are emerging as highly efficient vehicles for the delivery of genetic material. Herein, we review advances in gene delivery vectors targeting the liver and more specifically hepatocytes, covering strategies based on gene addition and gene editing, as well as the exciting results obtained with the use of RNA as a therapeutic molecule. Moreover, we will briefly summarise some of the limitations of current liver-directed gene therapy approaches and potential ways of overcoming them.

Ethylmalonic encephalopathy (EE) is a rare metabolic disorder caused by dysfunction of ETHE1 protein, a mitochondrial dioxygenase involved in hydrogen sulfide (H2S) detoxification. EE is usually a fatal disease with a severe clinical course mainly associated with developmental delay and regression, recurrent petechiae, orthostatic acrocyanosis, and chronic diarrhoea. Treatment includes antioxidants, antibiotics that lower H2S levels and antispastic medications, which are not curative. The mutations causing absence of the ETHE1 protein, as is the case for the described patient, usually entail a severe fatal phenotype. Although there are rare reported cases with mild clinical findings, the mechanism leading to these milder cases is also unclear. Here, we describe an 11-year-old boy with an ETHE1 gene mutation who has no neurocognitive impairment but chronic diarrhoea, which is controlled by oral medical treatment, and progressive spastic paraparesis that responded to Achilles tendon lengthening.

In the last years tandem mass spectrometry (MS/MS) has become a leading technology used for neonatal screening purposes. Newborn screening by MS/MS on dried blood spot samples (DBS) has one of its items in methionine levels: the knowledge of this parameter allows the identification of infant affected by homocystinuria (cystathionine β-synthase, CBS, deficiency) but can also lead, as side effect, to identify cases of methionine adenosyltransferase (MAT) type I/III deficiency.We started an expanded newborn screening for inborn errors of metabolism in Campania region in 2007. Here we report our ten years experience on expanded newborn screening in identifying patients affected by hypermethioninemia. During this period we screened approximately 77,000 infants and identified two cases: one case of classical homocystinuria and one patient affected by defect of MAT I/III. In this paper we describe these patients and their biochemical follow-up and review the literature concerning worldwide newborn screening reports on incidence of CBS and MAT deficiency. Keywords: Newborn screening, Hypermethioninemia, MAT I/III deficiency, CBS deficiency

Therapeutic strategies that increase hydrogen sulfide (H2S) or nitric oxide (NO) are cytoprotective in various models of cardiovascular injury. However, the nature of interaction between H2S and NO in heart failure and the underlying mechanisms for the protective effects remain undefined. The present study tested the cardioprotective effect of ZYZ-803, a novel synthetic H2S-NO hybrid molecule that decomposed to release H2S and NO. ZYZ-803 dose dependently improved left ventricular remodeling and preserved left ventricular function in the setting of isoprenaline-induced heart failure. The cardioprotective effect of ZYZ-803 is significantly more potent than that of H2S and/or NO donor alone. ZYZ-803 stimulated the expression of cystathionine γ-lyase (CSE) for H2S generation and the activity of endothelial NO synthase (eNOS) for NO production. Blocking CSE and/or eNOS suppressed ZYZ-803-induced H2S and NO production and cardioprotection. ZYZ-803 increased vascular endothelial growth factor (VEGF) concentration and cyclic guanosine 5′-monophosphate (cGMP) level. Moreover, ZYZ-803 upregulated the endogenous antioxidants, glutathione peroxidase (GPx) and heme oxygenase 1 (HO-1). These findings indicate that H2S and NO cooperatively attenuates left ventricular remodeling and dysfunction during the development of heart failure through VEGF/cGMP pathway and ZYZ-803 provide expanding insight into strategies for treatment of heart failure., Graphical abstract fx1

CBSs (cystathionine β-synthases) are eukaryotic PLP (pyridoxal 5 *-phosphate)-dependent proteins that maintain cellular homocysteine homoeostasis and produce cystathionine and hydrogen sulfide. In the present study, we describe a novel structural arrangement of the CBS enzyme encoded by the cbs-1 gene of the nematode Caenorhabditis elegans. The CBS-1 protein contains a unique tandem repeat of two evolutionarily conserved catalytic regions in a single polypeptide chain. These repeats include a catalytically active C-terminal module containing a PLP-binding site and a less conserved N-terminal module that is unable to bind the PLP cofactor and cannot catalyse CBS reactions, as demonstrated by analysis of truncated variants and active-site mutant proteins. In contrast with other metazoan enzymes, CBS-1 lacks the haem and regulatory Bateman domain essential for activation by AdoMet (S-adenosylmethionine) and only forms monomers. We determined the tissue and subcellular distribution of CBS-1 and showed that cbs-1 knockdown by RNA interference leads to delayed development and to an approximately 10-fold elevation of homocysteine concentrations in nematode extracts. The present study provides the first insight into the metabolism of sulfur amino acids and hydrogen sulfide in C. elegans and shows that nematode CBSs possess a structural feature that is unique among CBS proteins.

Non-alcoholic fatty liver disease (NAFLD) results from increased hepatic lipid accumulation and steatosis, and is closely linked to liver one-carbon (C1) metabolism. We assessed in C57BL6/N mice whether NAFLD induced by a high-fat (HF) diet over 8 weeks can be reversed by additional 4 weeks of a dietary methyl-donor supplementation (MDS). MDS in the obese mice failed to reverse NAFLD, but prevented the progression of hepatic steatosis associated with major changes in key hepatic C1-metabolites, e.g. S-adenosyl-methionine and S-adenosyl-homocysteine. Increased phosphorylation of AMPK-α together with enhanced β-HAD activity suggested an increased flux through fatty acid oxidation pathways. This was supported by concomitantly decreased hepatic free fatty acid and acyl-carnitines levels. Although HF diet changed the hepatic phospholipid pattern, MDS did not. Our findings suggest that dietary methyl-donors activate AMPK, a key enzyme in fatty acid β-oxidation control, that mediates increased fatty acid utilization and thereby prevents further hepatic lipid accumulation.