Biochemistry Unit

Barcelona, Spain

Biochemistry Unit

Barcelona, Spain
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Gregori J.,Vall dHebron Institute Recerca VHIR HUVH | Gregori J.,Hoffmann-La Roche | Gregori J.,University of Barcelona | Salicru M.,University of Barcelona | And 12 more authors.
Bioinformatics | Year: 2014

Given the inherent dynamics of a viral quasispecies, we are often interested in the comparison of diversity indices of sequential samples of a patient, or in the comparison of diversity indices of virus in groups of patients in a treated versus control design. It is then important to make sure that the diversity measures from each sample may be compared with no bias and within a consistent statistical framework. In the present report, we review some indices often used as measures for viral quasispecies complexity and provide means for statistical inference, applying procedures taken from the ecology field. In particular, we examine the Shannon entropy and the mutation frequency, and we discuss the appropriateness of different normalization methods of the Shannon entropy found in the literature. By taking amplicons ultra-deep pyrosequencing (UDPS) raw data as a surrogate of a real hepatitis C virus viral population, we study through in-silico sampling the statistical properties of these indices under two methods of viral quasispecies sampling, classical cloning followed by Sanger sequencing (CCSS) and next-generation sequencing (NGS) such as UDPS. We propose solutions specific to each of the two sampling methods-CCSS and NGS-to guarantee statistically conforming conclusions as free of bias as possible. © 2014 The Author.

Tugbobo O.S.,Biochemistry Unit | Adeniyi O.A.,Biochemistry Unit | Oyewusi H.A.,Biochemistry Unit
Der Pharmacia Lettre | Year: 2016

The antioxidant and toxicological effect of Allium sativum extract was investigated in rat liver in vitro. The effect of the antioxidant potential of the extract on 1,1-diphenyl-2-picryhydrazyl (DPPH) and hydroxyl radicals (OH*) was assessed while the toxicological effect of the extract on antioxidant enzymes such as reduced glutathione (GSH) and superoxide dismutase (SOD) as well as catalase (CAT) was also evaluated. The results show that aqueous and ethanolic extracts of Allium sativum cloves demonstrated significant (p<0.05) scavenging effect on DPPH and OH* radicals. It also indicates synergistic effect of the plant extract on antioxidant defense enzymes with concomitant increase in their activities compared to control. Therefore, Allium sativum could offer protection against free radical-induced oxidative stress and related diseases.

Davit-Spraul A.,Biochemistry Unit | Fabre M.,Pathology Unit | Fabre M.,University Paris - Sud | Branchereau S.,Pediatric Surgery Unit | And 10 more authors.
Hepatology | Year: 2010

Progressive familial intrahepatic cholestasis (PFIC) types 1 and 2 are characterized by normal serum gamma-glutamyl transferase (GGT) activity and are due to mutations in ATP8B1 (encoding FIC1) and ABCB11 (encoding bile salt export pump [BSEP]), respectively. Our goal was to evaluate the features that may distinguish PFIC1 from PFIC2 and ease their diagnosis. We retrospectively reviewed charts of 62 children with normal-GGT PFIC in whom a search for ATP8B1 and/or ABCB11 mutation, liver BSEP immunostaining, and/or bile analysis were performed. Based on genetic testing, 13 patients were PFIC1 and 39 PFIC2. The PFIC origin remained unknown in 10 cases. PFIC2 patients had a higher tendency to develop neonatal cholestasis. High serum alanine aminotransferase and alphafetoprotein levels, severe lobular lesions with giant hepatocytes, early liver failure, cholelithiasis, hepatocellular carcinoma, very low biliary bile acid concentration, and negative BSEP canalicular staining suggest PFIC2, whereas an absence of these signs and/or presence of extrahepatic manifestations suggest PFIC1. The PFIC1 and PFIC2 phenotypes were not clearly correlated with mutation types, but we found tendencies for a better prognosis and response to ursodeoxycholic acid (UDCA) or biliary diversion (BD) in a few children with missense mutations. Combination of UDCA, BD, and liver transplantation allowed 87% of normal-GGT PFIC patients to be alive at a median age of 10.5 years (1-36), half of them without liver transplantation. Conclusion: PFIC1 and PFIC2 differ clinically, biochemically, and histologically at presentation and/or during the disease course. A small proportion of normal-GGT PFIC is likely not due to ATP8B1 or ABCB11 mutations. Copyright © 2010 by the American Association for the Study of Liver Diseases.

This study assessed the nutrient, phytoconstituent, minerals, Zn bioavailability and antioxidant properties of mucilage extracted from Okra, Water leaf and Jews mallow. The protein in the mucilage of these vegetables was significantly higher (P ≤ 0.05) in water leaf (54.30%) than Jews mallow (44.80%) and okra (20.30%). Jews mallow had the highest fiber content (8.25%) and okra the lowest (2.00%). The calculated [Ca][phytate] / [Zn] molar ratios for the mucilage of water leaf (0.59) and Jews mallow (1.51) were clearly above the critical level of 0.5 mol / kg while that of the mucilage of okra (0.14) was below 0.5 mol / kg. The vitamin C content ranged from 5.00 mg AAE/g in okra to 10.25 mg AAE/g in water leaf. The result revealed that okra mucilage had a significantly higher (P ≤ 0.05) total phenol and reducing power than that of the mucilage of water leaf and Jews mallow. At the concentration of 100 and 300mg/ml, okra mucilage had the highest DPPH radical scavenging ability of 23.04% and 40.40% and the lowest OH radical scavenging ability of 13.16% and 59.03%. The mucilage of these vegetables could prevent protein malnutrition and serve as a natural antioxidant. © All Rights Reserved.

Hornig-Do H.T.,Northumbria University | Hornig-Do H.T.,University of Cologne | Montanari A.,University of Rome La Sapienza | Rozanska A.,Northumbria University | And 8 more authors.
EMBO Molecular Medicine | Year: 2014

Disorders of the mitochondrial genome cause a wide spectrum of disease, these present mainly as neurological and/or muscle related pathologies. Due to the intractability of the human mitochondrial genome there are currently no effective treatments for these disorders. The majority of the pathogenic mutations lie in the genes encoding mitochondrial tRNAs. Consequently, the biochemical deficiency is due to mitochondrial protein synthesis defects, which manifest as aberrant cellular respiration and ATP synthesis. It has previously been reported that overexpression of mitochondrial aminoacyl tRNA synthetases has been effective, in cell lines, at partially suppressing the defects resulting from mutations in their cognate mt-tRNAs. We now show that leucyl tRNA synthetase is able to partially rescue defects caused by mutations in non-cognate mt-tRNAs. Further, a C terminal peptide alone can enter mitochondria and interact with the same spectrum of mt-tRNAs as the entire synthetase, in intact cells. These data support the possibility that a small peptide could correct at least the biochemical defect associated with many mt-tRNA mutations, inferring a novel therapy for these disorders. © 2014 The Authors.

Lever M.,Biochemistry Unit | Slow S.,Biochemistry Unit
Clinical Biochemistry | Year: 2010

Betaine is an essential osmolyte and source of methyl groups and comes from either the diet or by the oxidation of choline. Its metabolism methylates homocysteine to methionine, also producing N,N-dimethylglycine. Betaine insufficiency is associated with the metabolic syndrome, lipid disorders and diabetes, and may have a role in vascular and other diseases. Betaine is important in development, from the pre-implantation embryo to infancy. Betaine supplementation improves animal and poultry health, but the effect of long-term supplementation on humans is not known, though reports that it improves athletic performance will stimulate further studies. Subsets of the population that may benefit from betaine supplementation could be identified by the laboratory, in particular those who excessively lose betaine through the urine.Plasma betaine is highly individual, in women typically 20-60μmol/L and in men 25-75μmol/L. Plasma dimethylglycine is typically <10μmol/L. Urine betaine excretion is minimal, even following a large betaine dose. It is constant, highly individual and normally <35. mmol/mole creatinine. The preferred method of betaine measurement is by LC-MS/MS, which is rapid and capable of automation. Slower HPLC methods give comparable results. Proton NMR spectrometry is another option but caution is needed to avoid confusion with trimethylamine-N-oxide. © 2010 The Canadian Society of Clinical Chemists.

Mackay R.J.,Biochemistry Unit | McEntyre C.J.,Biochemistry Unit | Henderson C.,Molecular Pathology Laboratory | Lever M.,Molecular Pathology Laboratory | And 2 more authors.
Clinical Biochemist Reviews | Year: 2011

Trimethylaminuria is a disorder in which the volatile, fish-smelling compound, trimethylamine (TMA) accumulates and is excreted in the urine, but is also found in the sweat and breath of these patients. Because many patients have associated body odours or halitosis, trimethylaminuria sufferers can meet serious difficulties in a social context, leading to other problems such as isolation and depression. TMA is formed by bacteria in the mammalian gut from reduction of compounds such as trimethylamine-N-oxide (TMAO) and choline. Primary trimethylaminuria sufferers have an inherited enzyme deficiency where TMA is not efficiently converted to the non-odorous TMAO in the liver. Secondary causes of trimethylaminuria have been described, sometimes accompanied by genetic variations. Diagnosis of trimethylaminuria requires the measurement of TMA and TMAO in urine, which should be collected after a high substrate meal in milder or intermittent cases, most simply, a marinefish meal. The symptoms of trimethylaminuria can be improved by changes in the diet to avoid precursors, in particular TMAO which is found in high concentrations in marine fish. Treatment with antibiotics to control bacteria in the gut, or activated charcoal to sequester TMA, may also be beneficial.

Lenky C.C.,University of Canterbury | McEntyre C.J.,University of Canterbury | McEntyre C.J.,Biochemistry Unit | Lever M.,University of Canterbury | Lever M.,Biochemistry Unit
Analytical Biochemistry | Year: 2012

Osmolytes are accumulated intracellularly to offset the effects of osmotic stress and protect cellular proteins against denaturation. Because different taxa accumulate different osmolytes, they can also be used as "dietary biomarkers" to study foraging. Potential osmolyte biomarkers include glycine betaine, trimethylamine N-oxide (TMAO), homarine, dimethylsulfoniopropionate (DMSP), and the osmolyte analog arsenobetaine (AsB). We present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous measurement of these osmolytes in serum or plasma. Varying concentrations of osmolytes were added to serum and samples and extracted in 90% acetonitrile and 10% methanol containing 10 μM deuterated internal standards (D 9-glycine betaine, D 9-trimethylamine-N-oxide, 13C 2-arsenobetaine, D 6-DMSP, and D 4-homarine). Analytes were separated on a normal-phase modified silica column and detected using isotope dilution tandem mass spectrometry in multiple reaction monitoring (MRM) mode. The assay was linear for all six compounds (r 2 values = 0.983-0.996). Recoveries were greater than 85%, and precision for within-batch coefficients of variation (CVs) were less than 8.2% and between-batch CVs were less than 6.1%. Limits of detection ranged from 0.02 to 0.12 μmol/L. LC-MS/MS is a simple method with high throughput for measuring low levels of osmolytes that are often present in biological samples. © 2011 Elsevier Inc. All rights reserved.

Davit-Spraul A.,Biochemistry Unit | Romdhane H.,Biochemistry Unit | Poggi-Bach J.,Biochemistry Unit
Journal of Chromatographic Science | Year: 2012

Tyrosinemia type 1, which is caused by a deficiency in fumarylacetoacetate hydrolase, is successfully treatable with nitisone (NTBC), an inhibitor of 4-hydroxyphenyl pyruvate dioxygenase. The recommended average dose of NTBC is 1 mg/kg per day. A rapid liquid chromatography (LC) coupled with negative electrospray ionization tandem mass spectrometry method was developed and validated for the quantification of NTBC in heparinized human plasma. The plasma samples were prepared by precipitation in acetonitrile. NTBC and the internal standard (IS) were chromatographed on a BEH C18 column. Gradient elution was done with a mixture of 10 mM ammonium acetate and methanol. The analyte was analyzed by LCtandem mass spectrometry with only 2 min run time. Selected reaction monitoring modes for detection of NTBC and the IS were achieved by using m/z 328 > 281 and 234 > 190, respectively. The LC retention times for NTBC and IS were 0.99 and 0.93 min, respectively. The method was linear in the concentration range of 0.75150 M with r < 0.998. Thus, this method is suitable for follow-up of patients treated with NTBC, because the current therapeutical concentrations range from 20 to 120 M. © 2012 The Author.

De Marco A.,Biochemistry Unit
Methods in Molecular Biology | Year: 2011

Molecular chaperones and chemical compounds like amino acids and osmolytes share the capability to prevent protein aggregation and can contribute to rescue in vivo aggregated proteins. Therefore, both overexpression of the molecular folding machinery and induced accumulation of chemical chaperones are options to improve the correct folding of recombinantly expressed proteins. These two parameters may show synergistic effects, although success remains protein specific and, therefore, several combinations of molecular and chemical chaperones should be compared. However, proteins can fail to fold correctly even in optimized culture conditions. In this case, protein aggregates can be recovered and their refolding assisted by an osmolyte/chaperone-dependent system. The selection of aggregates with different degrees of complexity can be exploited to maximize the yields of native proteins at the end of the refolding process. © 2011 Springer Science+Business Media, LLC.

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