APOB CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants

Hypobetalipoproteinemia is characterized by LDL-cholesterol and apolipoprotein B (apoB) plasma levels below the fifth percentile for age and sex. Familial hypobetalipoproteinemia (FHBL) is mostly caused by premature termination codons in the APOB gene, a condition associated with fatty liver and steatohepatitis. Nevertheless, many families with a FHBL phenotype carry APOB missense variants of uncertain significance (VUS). We here aimed to develop a proof-of-principle experiment to assess the pathogenicity of VUS using the genome editing of human liver cells. We identified a novel heterozygous APOB-VUS (p.Leu351Arg), in a FHBL family. We generated APOB knock-out (KO) and APOB-p.Leu351Arg knock-in Huh7 cells using CRISPR-Cas9 technology and studied the APOB expression, synthesis and secretion by digital droplet PCR and ELISA quantification. The APOB expression was decreased by 70% in the heterozygous APOB-KO cells and almost abolished in the homozygous-KO cells, with a consistent decrease in apoB production and secretion. The APOB-p.Leu351Arg homozygous cells presented with a 40% decreased APOB expression and undetectable apoB levels in cellular extracts and supernatant. Thus, the p.Leu351Arg affected the apoB secretion, which led us to classify this new variant as likely pathogenic and to set up a hepatic follow-up in this family. Therefore, the functional assessment of APOB-missense variants, using gene-editing technologies, will lead to improvements in the molecular diagnosis of FHBL and the personalized follow-up of these patients.     

Performance of Sulfate-reducing Passive Bioreactors for the Removal of Cd and Zn from Mine Drainage in a Cold Climate

Mine Water and the Environment - Passive treatment is a promising, green technology that is increasingly being used for mine drainage treatment. However, several challenges remain concerning its...     

Action Planning for Directed Model Checking of Petri Nets

Petri nets are fundamental to the analysis of distributed systems especially infinite-state systems. Finding a particular marking corresponding to a property violation in Petri nets can be reduced to exploring a state space induced by the set of reachable markings. Typical exploration(reachability analysis) approaches are undirected and do not take into account any knowledge about the structure of the Petri net. This paper proposes heuristic search for enhanced exploration to accelerate the search. For different needs in the system development process, we distinguish between different sorts of estimates.Treating the firing of a transition as an action applied to a set of predicates induced by the Petri net structure and markings, the reachability analysis can be reduced to finding a plan to an AI planning problem. Having such a reduction broadens the horizons for the application of AI heuristic search planning technology. In this paper we discuss the transformations schemes to encode Petri nets into PDDL. We show a concise encoding of general place-transition nets in Level 2 PDDL2.2, and a specification for bounded place-transition nets in ADL/STRIPS. Initial experiments with an existing planner are presented.     

Nucleoside diphosphate kinase. Investigation of the intersubunit contacts by site-directed mutagenesis and crystallography

NDP kinase from Dictyostelium was mutated by site-directed mutagenesis at positions indicated by structural data to be involved in the trimer interface. The mutants were substitutions at residue Pro-100 (P100S and P100G) and deletions of 1-5 residues at the C terminus. Single mutants yielded proteins that kept both activity and hexameric structure. However, they were severely affected in their stability toward temperature and urea denaturation. When the P100S mutation was combined with any of the C-terminal deletions, the enzyme lost most of its activity and dissociated into dimers. Crystallographic analysis of the P100S protein was performed at 2.6 A resolution. The x-ray structure showed no direct alteration of intersubunits contacts at residue 100, but an induced disruption of the interaction between Asp-115 and the C terminus of another subunit. The substitution of proline 100 to serine corresponds to the Killer-of-prune mutation in Drosophila. Consequences of the mutation are discussed in view of the structural and biochemical properties observed in the mutant Dictyostelium protein.     

Rational design of a triple helix-specific intercalating ligand

DNA triple helices offer new perspectives toward oligonucleotide-directed gene regulation. However, the poor stability of some of these structures might limit their use under physiological conditions. Specific ligands can intercalate into DNA triple helices and stabilize them. Molecular modeling and thermal denaturation experiments suggest that benzo[f]pyrido[3, 4-b]quinoxaline derivatives intercalate into triple helices by stacking preferentially with the Hoogsteen-paired bases. Based on this model, it was predicted that a benzo[f]quino[3,4-b]quinoxaline derivative, which possesses an additional aromatic ring, could engage additional stacking interactions with the pyrimidine strand of the Watson-Crick double helix upon binding of this pentacyclic ligand to a triplex structure. This compound was synthesized. Thermal denaturation experiments and inhibition of restriction enzyme cleavage show that this new compound can indeed stabilize triple helices with great efficiency and specificity and/or induce triple helix formation under physiological conditions.     

Influence of Organic Carbon Sources on Metal Removal from Mine Impacted Water Using Sulfate-Reducing Bacteria Bioreactors in Cold Climates

Mine Water and the Environment - Acid mine drainage (AMD) is a major environmental challenge for the mining industry in northern climates. Laboratory-scale experiments were conducted to test...     

Geometric Orbital Transfer Using Averaging Techniques

In this article, we focus on the coplanar orbital transfer of a satellite between Keplerian orbits, with low propulsion. We compute and analyze averaged systems associated with the energy minimization problem with the full control or control oriented in a single direction, tangential-normal or radial-orthoradial. This allows to understand the properties of each control direction in the optimal control problems.