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.     

Leakage radiation microscopy of surface plasmons launched by a nanodiamond-based tip

Leakage-radiation microscopy of a thin gold film demonstrates the ability of an ensemble of fluorescent diamond nanoparticles attached onto the apex of an optical tip to serve as an efficient near-field surface-plasmon polariton launcher. The implementation of the nanodiamond-based tip in a near-field scanning optical microscope will allow for an accurate control on the launching position, thereby opening the way to scanning plasmonics.     

Dynamic Control of Electron Transfers in Diflavin Reductases

Diflavin reductases are essential proteins capable of splitting the two-electron flux from reduced pyridine nucleotides to a variety of one electron acceptors. The primary sequence of diflavin reductases shows a conserved domain organization harboring two catalytic domains bound to the FAD and FMN flavins sandwiched by one or several non-catalytic domains. The catalytic domains are analogous to existing globular proteins: the FMN domain is analogous to flavodoxins while the FAD domain resembles ferredoxin reductases. The first structural determination of one member of the diflavin reductases family raised some questions about the architecture of the enzyme during catalysis: both FMN and FAD were in perfect position for interflavin transfers but the steric hindrance of the FAD domain rapidly prompted more complex hypotheses on the possible mechanisms for the electron transfer from FMN to external acceptors. Hypotheses of domain reorganization during catalysis in the context of the different members of this family were given by many groups during the past twenty years. This review will address the recent advances in various structural approaches that have highlighted specific dynamic features of diflavin reductases.     

Morphology of mucosa‐associated lymphoid tissue in odontocetes

This study describes the mucosa‐associated lymphoid tissue (MALT) in odontocetes from the Brazilian coast and freshwater systems. Seven species were evaluated and tissue samples were analyzed by light, scanning and transmission electron microscopy, and immunohistochemistry. Laryngeal tonsil was a palpable oval mass located in the larynx, composed of a lymphoepithelial complex. Dense collections of lymphocytes were found in the skin of male fetus and calf. Clusters of lymphoid tissue were found in the uterine cervix of a reproductively active juvenile female and along the pulmonary artery of an adult female. Lymphoid tissues associated with the gastrointestinal tract were characterized by diffusely arranged or organized lymphocytes. The anal tonsil was composed of an aggregate of lymphoid tissue occurring exclusively in the anal canal, being composed of squamous epithelium branches. MALT was present in different tissues and organic systems of cetaceans, providing constant protection against mucosal pathogens present in their environment.     

APOE Molecular Spectrum in a French Cohort with Primary Dyslipidemia

Primary hypercholesterolemia is characterized by elevated LDL-cholesterol (LDL-C) levels isolated in autosomal dominant hypercholesterolemia (ADH) or associated with elevated triglyceride levels in familial combined hyperlipidemia (FCHL). Rare APOE variants are known in ADH and FCHL. We explored the APOE molecular spectrum in a French ADH/FCHL cohort of 5743 unrelated probands. The sequencing of LDLR, PCSK9, APOB, and APOE revealed 76 carriers of a rare APOE variant, with no mutation in LDLR, PCSK9, or APOB. Among the 31 APOE variants identified here, 15 are described in ADH, 10 in FCHL, and 6 in both probands. Five were previously reported with dyslipidemia and 26 are novel, including 12 missense, 5 synonymous, 2 intronic, and 7 variants in regulatory regions. Sixteen variants were predicted as pathogenic or likely pathogenic, and their carriers had significantly lower polygenic risk scores (wPRS) than carriers of predicted benign variants. We observed no correlation between LDL-C levels and wPRS, suggesting a major effect of APOE variants. Carriers of p.Leu167del were associated with a severe phenotype. The analysis of 11 probands suggests that carriers of an APOE variant respond better to statins than carriers of a LDLR mutation. Altogether, we show that the APOE variants account for a significant contribution to ADH and FCHL.     

“Deterministic” quantum plasmonics

We demonstrate “deterministic” launching of propagative quantum surface-plasmon polaritons at freely chosen positions on gold plasmonic receptacles. This is achieved by using as a plasmon launcher a near-field scanning optical source made of a diamond nanocrystal with two nitrogen-vacancy color-center occupancy. Our demonstration relies on leakage-radiation microscopy of a thin homogeneous gold film and on near-field optical microscopy of a nanostructured thick gold film. Our work paves the way to future fundamental studies and applications in quantum plasmonics that require an accurate positioning of single-plasmon sources and may open a new branch in plasmonics and nanophotonics, namely scanning quantum plasmonics.     

Multiobjective optimization of MPLS-IP networks with a variable neighborhood genetic algorithm

This paper presents a Genetic Algorithm for the optimization of multiple indices of Quality of Service of Multi Protocol Label Switching (MPLS) IP networks. The proposed algorithm, the Variable Neighborhood Multiobjective Genetic Algorithm (VN-MGA), is a Genetic Algorithm based on the NSGA-II, with the particular feature that solutions are encoded defining two different kinds of neighborhoods. The first neighborhood is defined by considering as decision variables the edges that form the routes to be followed by each request, whilst the second part of solution is kept constant. The second neighborhood is defined by considering the request sequence as decision variable, with the first part kept constant. Comparisons are performed with: (i) a VNS algorithm that performs a switch between the same two neighborhoods that are used in VN-MGA; and (ii) the results obtained with an integer linear programming solver, running a scalarized version of the multiobjective problem. The results indicate that the proposed VN-MGA outperforms the pure VNS algorithm, and provides a good approximation of the exact Pareto fronts obtained with Integer Linear Programming (ILP) approach, at a much smaller computational cost. Besides potential benefits of the application of the proposed approach to the optimization of packet routing in MPLS networks, this work raises the theoretical issue of the systematic application of variable encodings, which allow variable neighborhood searches, as generic operators inside general evolutionary computation algorithms.     

Two-Phase Eutectic Growth in Al-Cu and Al-Cu-Ag

Metallurgical and Materials Transactions A - The microstructure developed by two-phase lamellar eutectics $$(\alpha )$$ – $$(\theta {\text {-Al}}_{2}{\text {Cu}})$$ in Al-Cu and Al-Cu-Ag...