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Papers by Ruben Ramos

Background Fecal microbiota transplantation (FMT) is an effective treatment of recurrent Clostrid... more Background Fecal microbiota transplantation (FMT) is an effective treatment of recurrent Clostridioides difficile (rCDI). In comparison FMT has more limited efficacy in treating either ulcerative colitis (UC) or Crohn's disease (CD), two forms of inflammatory bowel disesases (IBD). We hypothesize that FMT recipients with rCDI and/or IBD have baseline bile acid (BA)compositions that differ significantly from that of their healthy donorsand may be normalized by FMT. Aim To study the effect of single colonoscopic FMT on the microbial composition and function of recipients with rCDI and/or IBD. Methods Multi-omic analysis was performed on stools from 55 pairs of subjects and donors enrolled in two prospective single arm FMT clinical trials [ClinicalTrials.gov ID:NCT03268213, 479696, IND 15642, ClinicalTrials.gov ID: NCT03267238, IND 16795]. Fitted linear models were used to examine the effects of four recipient groups (rCDI withoutIBD, rCDI with IBD, UC without rCDI, CD without rCDI...

Blood, 2020

Studies of the relationship between the gastrointestinal microbiota and outcomes in allogeneic he... more Studies of the relationship between the gastrointestinal microbiota and outcomes in allogeneic hematopoietic stem cell transplantation (allo-HCT) have thus far largely focused on early complications, predominantly infection and acute graft-versus-host disease (GVHD). We examined the potential relationship of the microbiome with chronic GVHD (cGVHD) by analyzing stool and plasma samples collected late after allo-HCT using a case-control study design. We found lower circulating concentrations of the microbe-derived short-chain fatty acids (SCFAs) propionate and butyrate in day 100 plasma samples from patients who developed cGVHD, compared with those who remained free of this complication, in the initial case-control cohort of transplant patients and in a further cross-sectional cohort from an independent transplant center. An additional cross-sectional patient cohort from a third transplant center was analyzed; however, serum (rather than plasma) was available, and the differences in ...

Nature, 2019

C. and V.E. cultured bacterial isolates from fecal samples and analyzed whole genome sequences of... more C. and V.E. cultured bacterial isolates from fecal samples and analyzed whole genome sequences of isolates. P.V.S. and R.C.H. performed peptide purifications and subsequent characterization by mass spectrometry. E.R.L. performed bioinformatic analyses and metagenomic sequence data. R.S. assisted in bacterial culturing and animal experiments. I.M.L. and R.S. maintained and screened mouse strains. MG generated and characterized bacterial isolates from fecal samples. W.Q., R.J.J.F.R. and J.R.C. contributed to development of methods to purify bacterial lantibiotics for biochemical analyses. E.F., L.A. and R.W. performed DNA extractions, 16S MiSeq Illumina sequencing and analyzed microbiome sequence data. Z.-M.X.W. assisted in ileal homogenization, western blot, and RT-qPCR analyses. H.-J.J. contributed to the cloning and expression of the lantibiotic gene. S.M.M. and Y.T. enrolled patients undergoing allogeneic hematopoietic cell transplantation in the prospective fecal collection protocol and contributed to the analysis of sequence data. S.N. and K.H. contributed human-derived commensal bacterial strains that were included in this study. J.U.P. and M.R.M.v.d.B. contributed to the analyses of patient-derived fecal samples.

The American Journal of Human Genetics, 2019

Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate d... more Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects.

Human molecular genetics, Jan 18, 2018

Loss-of-function mutations in glutaminase (GLS), the enzyme converting glutamine into glutamate, ... more Loss-of-function mutations in glutaminase (GLS), the enzyme converting glutamine into glutamate, and the counteracting enzyme glutamine synthetase (GS) cause disturbed glutamate homeostasis and severe neonatal encephalopathy. We report a de novo Ser482Cys gain-of-function variant in GLS encoding glutaminase associated with profound developmental delay and infantile cataract. Functional analysis demonstrated that this variant causes hyperactivity and compensatory downregulation of GLS expression combined with upregulation of the counteracting enzyme GS, supporting pathogenicity. Ser482Cys-GLS likely improves the electrostatic environment of the GLS catalytic site, thereby intrinsically inducing hyperactivity. Alignment of +/-12.000 GLS protein sequences from >1000 genera, revealed extreme conservation of Ser482, to the same degree as catalytic residues. Together with the hyperactivity, this indicates that Ser482 is evolutionarily preserved to achieve optimal -but submaximal- GLS a...

Journal of Inherited Metabolic Disease, 2017

Pyridoxal 5′-phosphate (PLP), the metabolically active form of vitamin B6, plays an essential rol... more Pyridoxal 5′-phosphate (PLP), the metabolically active form of vitamin B6, plays an essential role in brain metabolism as a cofactor in numerous enzyme reactions. PLP deficiency in brain, either genetic or acquired, results in severe drug-resistant seizures that respond to vitamin B6 supplementation. The pathogenesis of vitamin B6 deficiency is largely unknown. To shed more light on the metabolic consequences of vitamin B6 deficiency in brain, we performed untargeted metabolomics in vitamin B6-deprived Neuro-2a cells. Significant alterations were observed in a range of metabolites. The most surprising observation was a decrease of serine and glycine, two amino acids that are known to be elevated in the plasma of vitamin B6 deficient patients. To investigate the cause of the low concentrations of serine and glycine, a metabolic flux analysis on serine biosynthesis was performed. The metabolic flux results showed that the de novo synthesis of serine was significantly reduced in vitamin B6deprived cells. In addition, formation of glycine and 5methyltetrahydrofolate was decreased. Thus, vitamin B6 is essential for serine de novo biosynthesis in neuronal cells, and serine de novo synthesis is critical to maintain intracellular serine and glycine. These findings suggest that serine and glycine concentrations in brain may be deficient in patients with vitamin B6 responsive epilepsy. The low intracellular 5-mTHF concentrations observed in vitro may explain the favourable but so far unexplained response of some patients with pyridoxine-dependent epilepsy to folinic acid supplementation.

Gastroenterology, 2017

BACKGROUND & AIMS: The nuclear receptor subfamily 1 group H member 4 (NR1H4 or farnesoid X recept... more BACKGROUND & AIMS: The nuclear receptor subfamily 1 group H member 4 (NR1H4 or farnesoid X receptor [FXR]) regulates bile acid synthesis, transport, and catabolism. FXR also regulates postprandial lipid and glucose metabolism. We performed quantitative proteomic analyses of liver tissues from mice to evaluate these functions and investigate whether FXR regulates amino acid metabolism. METHODS: To study the role of FXR in mouse liver, we used mice with a disruption of Nr1h4 (FXR-knockout mice) and compared them with floxed control mice. Mice were gavaged with the FXR agonist obeticholic acid or vehicle for 11 days. Proteome analyses, as well as targeted metabolomics and chromatin immunoprecipitation, were performed on the livers of these mice. Primary rat hepatocytes were used to validate the role of FXR in amino acid catabolism by gene expression and metabolomics studies. Finally, control mice and mice with liver-specific disruption of Nr1h4 (liver FXR-knockout mice) were re-fed with a highprotein diet after 6 hours fasting and gavaged a 15 NH 4 Cl tracer. Gene expression and the metabolome were studied in the livers and plasma from these mice. RESULTS: In livers of control mice and primary rat hepatocytes, activation of FXR with obeticholic acid increased expression of proteins that regulate amino acid degradation, ureagenesis, and glutamine synthesis. We found FXR to bind to regulatory sites of genes encoding these proteins in control livers. Liver tissues from FXR-knockout mice had reduced expression of urea cycle proteins, and accumulated precursors of ureagenesis, compared with control mice. In liver FXR-knockout mice on a high-protein diet, the plasma concentration of newly formed urea was significantly decreased compared with controls. In addition, liver FXR-knockout mice had reduced hepatic expression of enzymes that regulate ammonium detoxification compared with controls. In contrast, obeticholic acid increased expression of genes encoding enzymes involved in ureagenesis compared with vehicle in C57Bl/6 mice. CONCLUSIONS: In livers of mice, FXR regulates amino acid catabolism and detoxification of ammonium via ureagenesis and glutamine synthesis. Failure of the urea cycle and hyperammonemia are common in patients with acute and chronic liver diseases; compounds that activate FXR might promote ammonium clearance in these patients.

Manganese (Mn) is an essential element and it acts as a cofactor for a number of enzymatic reacti... more Manganese (Mn) is an essential element and it acts as a cofactor for a number of enzymatic reactions, including those involved in amino acid, lipid, protein and carbohydrate metabolism. Excessive exposure to Mn can lead to poisoning, characterized by psychiatric disturbances and an extrapyramidal disorder. Mn-induced neuronal degeneration is associated with alterations in amino acids metabolism. In the present study, we analyzed whole rat brain amino acid content subsequent to 4 or 8 intraperitoneal (ip) injections, with 25 mg MnCl 2 /kg/day, at 48-hour (h) intervals. We noted a significant increase in glycine brain levels after 4 or 8 Mn injections (p<0.05 and p<0.01, respectively) and arginine also after 4 or 8 injections (p<0.001). Significant increases were also noted in brain proline (p<0.01), cysteine (p<0.05), phenylalanine (p<0.01) and tyrosine (p<0.01) levels after 8 Mn injections vs. the control group. These findings suggest that Mninduced alterations in amino acid levels secondary to Mn affect the neurochemical milieu. Index Entries neurotoxicity; brain; manganese; γ-aminobutyric acid (GABA); glutamate; amino acids

Journal of Inherited Metabolic Disease, 2015

We present the first two reported unrelated patients with an isolated sedoheptulokinase (SHPK) de... more We present the first two reported unrelated patients with an isolated sedoheptulokinase (SHPK) deficiency. The first patient presented with neonatal cholestasis, hypoglycemia, and anemia, while the second patient presented with congenital arthrogryposis multiplex, multiple contractures, and dysmorphisms. Both patients had elevated excretion of erythritol and sedoheptulose, and each had a homozygous nonsense mutation in SHPK. SHPK is an enzyme that phosphorylates sedoheptulose to sedoheptulose‐7‐phosphate, which is an important intermediate of the pentose phosphate pathway. It is questionable whether SHPK deficiency is a causal factor for the clinical phenotypes of our patients. This study illustrates the necessity of extensive functional and clinical workup for interpreting a novel variant, including nonsense variants.

Sumário xiii Chapter 1 Aims and outline of the thesis 1 Chapter 2 General introduction 7 Chapter ... more Sumário xiii Chapter 1 Aims and outline of the thesis 1 Chapter 2 General introduction 7 Chapter 3 Gas-Chromatography (GC) analysis of sugars and polyols: optimization of a GC-FID and a GC-MS method 37 Chapter 4 Pentose phosphate pathway intermediates: a study in adult patients with liver dysfunction 69 Chapter 5 Pentose phosphate pathway intermediates involved in liver dysfunction: a study in a pediatric group 83 Chapter 6 Final remarks and perspectives 97 Acknowledgments/Agradecimentos

Molecular genetics & genomic medicine, 2014

Galactose-1-phosphate uridylyltransferase (GALT) is a key enzyme in galactose metabolism, particu... more Galactose-1-phosphate uridylyltransferase (GALT) is a key enzyme in galactose metabolism, particularly important in the neonatal period due to ingestion of galactose-containing milk. GALT deficiency results in the genetic disorder classic galactosemia, whose pathophysiology is still not fully elucidated. Whereas classic galactosemia has been hypothesized to result from GALT misfolding, a thorough functional-structural characterization of GALT most prevalent variants was still lacking, hampering the development of alternative therapeutic approaches. The aim of this study was to investigate the structural-functional effects of nine GALT mutations, four of which account for the vast majority of the mutations identified in galactosemic patients. Several methodologies were employed to evaluate the mutations' impact on GALT function, on the protein secondary and tertiary structures, and on the aggregation propensity. The major structural effect concerns disturbed propensity for aggreg...

Journal of Inherited Metabolic Disease, 2013

Classic galactosemia is an autosomal recessive disorder caused by deficient galactose‐1‐phosphate... more Classic galactosemia is an autosomal recessive disorder caused by deficient galactose‐1‐phosphate uridylyltransferase (GALT) activity. Patients develop symptoms in the neonatal period, which can be ameliorated by dietary restriction of galactose. Many patients develop long‐term complications, with a broad range of clinical symptoms whose pathophysiology is poorly understood. The high allelic heterogeneity of GALT gene that characterizes this disorder is thought to play a determinant role in biochemical and clinical phenotypes. We aimed to characterize the mutational spectrum of GALT deficiency in Portugal and to assess potential genotype‐phenotype correlations. Direct sequencing of the GALT gene and in silico analyses were employed to evaluate the impact of uncharacterized mutations upon GALT functionality. Molecular characterization of 42 galactosemic Portuguese patients revealed a mutational spectrum comprising 14 nucleotide substitutions: ten missense, two nonsense and two putati...

Clinical Biochemistry, 2010

Vitamin B(12), or B(12), is an essential nutrient for humans, and its deficiency is a public heal... more Vitamin B(12), or B(12), is an essential nutrient for humans, and its deficiency is a public health problem, especially in elderly population. Around 30% of circulating total B(12) levels are attached to transcobalamin II (TCN2), being referred as holotranscobalamin (holo-TC), and representing the biologically active fraction. After cellular uptake, B(12) participates in the homocysteine (Hcy) metabolism. The potential influence of the described TCN2 776CNG polymorphism upon B(12) intracellular delivery is a current target of research and we aimed to investigate its biochemical significance upon a healthy adult population.

Molecular Genetics and Metabolism, 2011

Hyperphenylalaninemia (HPA, OMIM #261600), which includes phenylketonuria (PKU), is caused by mut... more Hyperphenylalaninemia (HPA, OMIM #261600), which includes phenylketonuria (PKU), is caused by mutations in the gene encoding phenylalanine hydroxylase (PAH), being already described more than 600 different mutations. Genotype-phenotype correlation is a useful tool to predict the metabolic phenotype, to establish the better tailored diet and, more recently, to assess the potential responsiveness to BH 4 therapy, a current theme on PKU field. The aim of this study was the molecular analysis of the PAH gene, evaluation of genotype-phenotype relationships and prediction of BH 4-responsiveness in the HPA population living in South Portugal. We performed the molecular characterization of 83 HPA patients using genomic DNA extracted from peripheral blood samples or Guthrie cards. PAH mutations were scanned by PCR amplification of exons and related intronic boundaries, followed by direct sequence analysis. Intragenic polymorphisms were determined by PCR-RFLP analysis. The results allowed the full characterization of 67 patients. The mutational spectrum encompasses 34 distinct mutations, being the most frequent IVS10nt-11GNA (14.6%), V388M (10.8%), R261Q (8.2%) and R270K (7.6%), which account for 46% of all mutant alleles. Moreover, 12 different haplotypes were identified and most mutations were associated with a single one. Notably, more than half of the 34 mutations belong to the group of more than 70 mutations already identified in BH 4-responsive patients, according to BIOPKU database. Fifty one different genotypic combinations were found, most of them in single patients and involving a BH 4responsive mutation. In conclusion, a significant number (30-35%) of South Portugal PKU patients may potentially benefit from BH 4 therapy which, combined with a less strict diet, or eventually in special cases as monotherapy, may contribute to reduce nutritional deficiencies and minimize neurological and psychological dysfunctions.