Mobile Element Insertion in the APOB Exon 3 Coding Sequence: A New Challenge in Hypobetalipoproteinemia Diagnosis.

Mobile elements (ME) can transpose by copy-and-paste mechanisms. A heterozygous insertion in APOB exon 3 coding sequence was suspected in a patient with hypobetalipoproteinemia (HBL), by gel electrophoresis of the PCR products. An insertion of a 85 bp fragment flanked by a polyA stretch and a target replication site duplication was identified as a ME insertion (MEI) from the AluYa5 subfamily, NM_000384.

Immobilization of Enzymes on Magnetic Beads Through Affinity Interactions.

The development of enzyme immobilization techniques that will not affect catalytic activity and conformation is an important research task. Affinity tags that are present or added at a specific position far from the active site in the structure of the native enzyme could be used to create strong affinity bonds between the protein structure and a surface functionalized with the complementary affinity ligand. These immobilization techniques are based on affinity interactions between biotin and (strept)avidin molecules, lectins and sugars, or metal chelate and histidine tag.

Efficacy of two vitamin E formulations in patients with abetalipoproteinemia and chylomicron retention disease.

Abetalipoproteinemia (ABL) and chylomicron retention disease (CMRD) are extremely rare recessive forms of hypobetalipoproteinemia characterized by intestinal lipid malabsorption and severe vitamin E deficiency. Vitamin E is often supplemented in the form of fat-soluble vitamin E acetate, but fat malabsorption considerably limits correction of the deficiency. In this crossover study, we administered two different forms of vitamin E, tocofersolan (a water-soluble derivative of RRR-α-tocopherol) and α-tocopherol acetate, to three patients with ABL and four patients with CMRD.

Homozygous familial hypercholesterolemia in childhood: Genotype-phenotype description, established therapies and perspectives.

Familial hypercholesterolemia (FH) is a co-dominantly inherited disorder of plasma lipoprotein metabolism. The prevalence of heterozygous FH (HeFH) is between 1/500 and 1/200 whereas that of homozygous form (HoFH) is about 1/1,000,000. Diagnosis is based on cutaneous xanthomas and untreated levels of LDL-cholesterol over 500 mg/dl before 10 years of age.

Lipid profile and cardiovascular risk factors in pediatric liver transplant recipients.

Cardiovascular diseases induce long-term morbidity and mortality of adult LT recipients. The aim of this retrospective study was to assess CVRF, lipid abnormalities, and atherosclerosis (appraised by c-IMT), more than 10 yr after pediatric LT. Thirty-one children who underwent LT between December 1990 and December 2000 were included.

Polyluminol/hydrogel composites as new electrochemiluminescent-active sensing layers.

This paper reports on electrochemiluminescent sensors and biosensors based on polyluminol/hydrogel composite sensing layers using chemical or biological membranes as hydrogel matrices. In this work, luminol is electropolymerized under near-neutral conditions onto screen-printed electrode (SPE)-supported hydrogel films. The working electrode coated with a hydrogel film is soaked in a solution containing monomeric luminol units, allowing the monomeric luminol units to diffuse inside the porous matrix to the electrode surface where they are electropolymerized by cyclic voltammetry (CV).

Post-heparin LPL activity measurement using VLDL as a substrate: a new robust method for routine assessment of plasma triglyceride lipolysis defects.

Background: Determination of lipoprotein lipase (LPL) activity is important for hyperchylomicronemia diagnosis, but remains both unreliable and cumbersome with current methods. Consequently by using human VLDL as substrate we developed a new LPL assay which does not require sonication, radioactive or fluorescent particles.

Methods: Post-heparin plasma was added to the VLDL substrate prepared by ultracentrifugation of heat inactivated normolipidemic human serums, diluted in buffer, pH 8.

Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia.

Background & Aims: Non-alcoholic steatohepatitis leading to fibrosis occurs in patients with abetalipoproteinemia (ABL) and homozygous or compound heterozygous familial hypobetalipoproteinemia (Ho-FHBL). We wanted to establish if liver alterations were more frequent in one of both diseases and were influenced by comorbidities.

Methods: We report genetic, clinical, histological and biological characteristics of new cases of ABL (n =7) and Ho-FHBL (n = 7), and compare them with all published ABL (51) and Ho-FHBL (22) probands.

Post-heparin LPL activity measurement using VLDL as a substrate: a new robust method for routine assessment of plasma triglyceride lipolysis defects.

Background: Determination of lipoprotein lipase (LPL) activity is important for hyperchylomicronemia diagnosis, but remains both unreliable and cumbersome with current methods. Consequently by using human VLDL as substrate we developed a new LPL assay which does not require sonication, radioactive or fluorescent particles.

Methods: Post-heparin plasma was added to the VLDL substrate prepared by ultracentrifugation of heat inactivated normolipidemic human serums, diluted in buffer, pH 8.

An APOA5 3' UTR variant associated with plasma triglycerides triggers APOA5 downregulation by creating a functional miR-485-5p binding site.

APOA5 c.*158C>T (rs2266788), located in the 3' UTR, belongs to APOA5 haplotype 2 (APOA5*2), which is strongly associated with plasma triglyceride levels and modulates the occurrence of both moderate and severe hypertriglyceridemia. Individuals with APOA5*2 display reduced APOA5 expression at the posttranscriptional level.

Enzyme immobilization by entrapment within a gel network.

This chapter provides a detailed description of the three immobilization methods based on the biomolecules entrapment into polymer matrices. The poly (vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ), a soluble pre-polymer bearing photo-cross-linkable groups, has widely been used to entrap enzymes, and several bioassays based on this immobilization matrix have been reported. Similarly, immobilization of enzymes via sol-gel has been described in this chapter.

Immobilization of enzymes on ethynyl-modified electrodes via click chemistry.

This paper describes a novel, simple, and versatile protocol for covalent immobilization of enzyme on electrode. The immobilization method is based on the combination of diazonium salt electrografting and click chemistry. The ethynyl-terminated monolayers are obtained by diazonium salt electrografting, then, in the presence of copper (I) catalyst, the ethynyl modified surfaces reacts efficiently and rapidly with enzyme bearing an azide function (azido-enzyme), thus forming a covalent 1,2,3-triazole linkage by means of click chemistry.

Immobilization of enzymes on magnetic beads through affinity interactions.

The development of enzyme immobilization techniques that will not affect catalytic activity and conformation is an important research task. Affinity tags that are present or added at a specific position far from the active site in the structure of the native enzyme could be used to create strong affinity bonds between the protein structure and a surface functionalized with the complementary affinity ligand. These immobilization techniques are based on affinity interactions between biotin and (strept)avidin molecules, lectins and sugars, or metal chelate and histidine tag.

Detection of the marine toxin okadaic acid: assessing seafood safety.

Diarrheic Shellfish Poisoning (DSP) is a gastrointestinal illness caused by consumption of shellfish contaminated with DSP toxins such as okadaic acid (OA) and dinophysistoxins (DTX). The occurrences of OA in bivalves induce not only public health problems but also economic damages to shellfish farming. Consequently, the development of fast, reliable and sensitive detection methods is an evident necessity.

Molecular characterization of Tunisian families with abetalipoproteinemia and identification of a novel mutation in MTTP gene.

Background: Abetalipoproteinemia (ABL; OMIM 200100) is a rare monogenic disorder of lipid metabolism characterized by reduced plasma levels of total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C) and almost complete absence of apolipoprotein B (apoB). ABL results from genetic deficiency in microsomal triglyceride transfer protein (MTP; OMIM 157147). In the present study we investigated two unrelated Tunisian patients, born from consanguineous marriages, with severe deficiency of plasma low-density lipoprotein (LDL) and apo B.

Inhibitory effects of in vivo oxidized high-density lipoproteins on platelet aggregation: evidence from patients with abetalipoproteinemia.

There is evidence that high-density lipoproteins (HDLs) may regulate platelet function, but disparate results exist regarding the effects of oxidized HDLs on platelets. The objective of our study was to determine the role of in vivo oxidized HDLs on platelet aggregation. Platelet aggregation and redox status were investigated in 5 patients with abetalipoproteinemia (ABLP) or homozygous hypobetalipoproteinemia, two rare metabolic diseases characterized by the absence of apolipoprotein B-containing lipoproteins, compared to 5 control subjects.

Chylomicron retention disease: report of two cases from a Greek Island.

Chylomicron retention disease (CRD), or Anderson disease, is a rare, hereditary cause of fat malabsorption. It is one of the familial hypocholesterolaemia syndromes, along with homozygous hypobetalipoproteinaemia (HBL) and abetalipoproteinaemia (ABL). We report clinical, laboratory and histological data as well as molecular DNA analysis in the case of a 4-month-old boy with failure to thrive and steatorrhea who was diagnosed with CRD.

Highly sensitive ochratoxin A impedimetric aptasensor based on the immobilization of azido-aptamer onto electrografted binary film via click chemistry.

The aptamer immobilization onto organized mixed layers of diazonium salts via click chemistry was explored. The immobilized aptamer was employed in the fabrication of a highly sensitive and reusable electrochemical impedimetric aptasensor for the detection of ochratoxin A (OTA). The screen-printed carbon electrodes (SPCEs) were first modified by electrografting of a protected 4-((trimethylsilyl)ethynyl) benzene (TMSi-Eth-Ar) layer followed by a second one of p-nitrobenzene (p-NO(2)-Ar) by means of electrochemical reduction of their corresponding diazonium salts, (TMSi-Eth-Ar-N(2)(+)) and (p-NO(2)-ArN(2)(+)).

Automated flow-through amperometric immunosensor for highly sensitive and on-line detection of okadaic acid in mussel sample.

An electrochemical immunosensor for okadaic acid (OA) detection has been developed, and used in an indirect competitive immunoassay format under automated flow conditions. The biosensor was fabricated by injecting OA modified magnetic beads onto screen printed carbon electrode (SPCE) in the flow system. The OA present in the sample competed with the immobilized OA to bind with anti-okadaic acid monoclonal antibody (anti-OA-MAb).

Highly sensitive detection and discrimination of LR and YR microcystins based on protein phosphatases and an artificial neural network.

The inhibition characteristics of three different protein phosphatases by three microcystin (MC) variants--LR, YR, and RR--were studied. The corresponding K (I) for each enzyme-MC couple was calculated. The toxicity of MC varies in the following order: MC-LR > MC-YR > MC-RR.

Development of a novel label-free amperometric immunosensor for the detection of okadaic acid.

Okadaic acid (OA), a lipophilic phycotoxin is mainly produced by toxigenic dinoflagellates. The need to develop high performing methods for OA analysis able to improve the traditional ones is evident. In this work, a novel experimental methodology for label-free detection of OA was developed.

Molecular and functional analysis of two new MTTP gene mutations in an atypical case of abetalipoproteinemia.

Abetalipoproteinemia (ABL) is an inherited disease characterized by the defective assembly and secretion of apolipoprotein B-containing lipoproteins caused by mutations in the microsomal triglyceride transfer protein large subunit (MTP) gene (MTTP). We report here a female patient with an unusual clinical and biochemical ABL phenotype. She presented with severe liver injury, low levels of LDL-cholesterol, and subnormal levels of vitamin E, but only mild fat malabsorption and no retinitis pigmentosa or acanthocytosis.

Clinical and electrophysiological characteristics of neuropathy associated with Tangier disease.

Tangier disease (TD) (OMIM#205400) is a rare autosomal recessive disorder resulting from mutations in the ABCA1 gene, leading to decreased levels of plasma high-density lipoproteins (HDL). Peripheral neuropathy is a common finding in this disease, and may present as relapsing/remitting mono/polyneuropathies or as syringomyelia-like neuropathy. We retrospectively analyzed four patients, and report here their clinical, biological, electrophysiological, imaging, and genetic findings.

Development of a colorimetric inhibition assay for microcystin-LR detection: comparison of the sensitivity of different protein phosphatases.

A colorimetric protein phosphatase (PP) inhibition test for the detection of microcystin-LR (MC-LR) has been developed. Three PP2As, one recombinant and two natural versions, as well as one PP1 produced by molecular engineering, were tested. First, assays were performed using the enzymes in solution to compare their sensitivity to MC-LR.

Immobilization strategies to develop enzymatic biosensors.

Immobilization of enzymes on the transducer surface is a necessary and critical step in the design of biosensors. An overview of the different immobilization techniques reported in the literature is given, dealing with classical adsorption, covalent bonds, entrapment, cross-linking or affinity as well as combination of them and focusing on new original methods as well as the recent introduction of promising nanomaterials such as conducting polymer nanowires, carbon nanotubes or nanoparticles. As indicated in this review, various immobilization methods have been used to develop optical, electrochemical or gravimetric enzymatic biosensors.