Apolipoprotein E,a Crucial Cellular Protein in the Lifecycle of Hepatitis Viruses

Apolipoprotein E (ApoE) is a multifunctional protein expressed in several tissues, including those of the liver. This lipoprotein component is responsible for maintaining lipid content homeostasis at the plasma and tissue levels by transporting lipids between the liver and peripheral tissues. The ability of ApoE to interact with host-cell surface receptors and its involvement in several cellular pathways raised questions about the hijacking of ApoE by hepatotropic viruses. Hepatitis C virus (HCV) was the first hepatitis virus reported to be dependent on ApoE for the completion of its lifecycle, with ApoE being part of the viral particle, mediating its entry into host cells and contributing to viral morphogenesis. Recent studies of the hepatitis B virus (HBV) lifecycle have revealed that this virus and its subviral envelope particles also incorporate ApoE. ApoE favors HBV entry and is crucial for the morphogenesis of infectious particles, through its interaction with HBV envelope glycoproteins. This review summarizes the data highlighting the crucial role of ApoE in the lifecycles of HBV and HCV and discusses its potential role in the lifecycle of other hepatotropic viruses.     

Myelinosome Organelles in the Retina of R6/1 Huntington Disease (HD) Mice: Ubiquitous Distribution and Possible Role in Disease Spreading

Visual deficit is one of the complications of Huntington disease (HD), a fatal neurological disorder caused by CAG trinucleotide expansions in the Huntingtin gene, leading to the production of mutant Huntingtin (mHTT) protein. Transgenic HD R6/1 mice expressing human HTT exon1 with 115 CAG repeats recapitulate major features of the human pathology and exhibit a degeneration of the retina. Our aim was to gain insight into the ultrastructure of the pathological HD R6/1 retina by electron microscopy (EM). We show that the HD R6/1 retina is enriched with unusual organelles myelinosomes, produced by retinal neurons and glia. Myelinosomes are present in all nuclear and plexiform layers, in the synaptic terminals of photoreceptors, in the processes of retinal neurons and glial cells, and in the subretinal space. In vitro study shows that myelinosomes secreted by human retinal glial Müller MIO-M1 cells transfected with EGFP-mHTT-exon1 carry EGFP-mHTT-exon1 protein, as revealed by immuno-EM and Western-blotting. Myelinosomes loaded with mHTT-exon1 are incorporated by naive neuronal/neuroblastoma SH-SY5Y cells. This results in the emergence of mHTT-exon1 in recipient cells. This process is blocked by membrane fusion inhibitor MDL 28170. Conclusion: Incorporation of myelinosomes carrying mHTT-exon1 in recipient cells may contribute to HD spreading in the retina. Exploring ocular fluids for myelinosome presence could bring an additional biomarker for HD diagnostics.     

Preparation of Spherical, Submicrometer Oxide Particles by Hydrolysis of Emulsified Alkoxide Droplets

A new method for forming spherical, submicrometer ceramic oxide particles by the hydrolysis of emulsified alkoxide droplets is reported. Emulsions are formed of alkoxide droplets dispersed in an inert, polar solvent. The alkoxide droplets are hydrolyzed to form oxide particles by adding water to the emulsion. It was shown that individual droplets acted as "microreactors" and controlled the powder size, shape, and composition. Both single-oxide and mixed-oxide powders were formed by this technique.     

In vitro comparison of hepatic metabolism of 9cis-11 trans and 10trans-12cis isomers of CLA in the rat

Hepatic metabolism of the two main isomers of CLA (9cis-11 trans, 10trans-12cisC_18∶2) was compared to that of oleic acid (representative of the main plasma FA) in 16 rats by using the in vitro method of incubated liver slices. Liver tissue samples were incubated at 37°C for 17h under an atmosphere of 95% O₂/5%CO₂ in a medium supplemented with 0.75 mM of FA mixture (representative of circulating nonesterified FA) and with 55 μM [1-¹⁴C]9cis-11 trans C_18∶2, [1-¹⁴C]10trans-12cis C_18∶2, or [1-¹⁴C]oleate. The uptake of CLA by hepatocytes was similar for both isomers (9%) and was three times higher (P<0.01) than for oleate (2.6%). The rate of CLA isomer oxidation was two times higher (49 and 40% of incorporated amounts of 9cis-11 trans and 10trans-12 cis, respectively) than that of oleate (P<0.01). Total oxidation of oleate and CLA isomers into [¹⁴CO₂] was low (2 to 7% of total oxidized FA) compared to the partial oxidation (93 to 98%) leading to the production of [¹⁴C] acid-soluble products. CLA isoemrs escaping from catabolism were both highly desaturated (26.7 and 26.8%) into conjugated 18∶3. Oleate and CLA isomers were mainly esterified into neutral lipids (30%). They were slowly secreted as parts of VLDL particles (<0.4% of FA incorporated into cells), the extent of secretion of oleate and of 10trans-12 cis being 2.2-fold higher than that of 9cis-11 trans (P<0.02). In conclusion, this study clearly showed that both CLA isomers were highly catabolized by hepatocytes, reducing their availability for peripheral tissues. Moreover, more than 25% of CLA escaping from catabolism was converted into conjugated 18∶3, the biological properties of which remain to be elucidated.     

Tamoxifen derivatives for delivery of the antitumoral (DACH)Pt group: selective synthesis by McMurry coupling,and biochemical behaviour

The goal of our study was to potentiate the effects of the ((R,R)-trans-1,2-diaminocyclohexane)-platinum(II) fragment [(DACH)Pt], known for its cytotoxic properties, either with tamoxifen (Tam), the most widely used antiestrogen in the treatment of hormone-dependent breast cancers, or with its active metabolite hydroxytamoxifen (hydroxy-Tam). We coupled Tam or hydroxy-Tam derivatives bearing a malonato group at the para position of the beta aromatic ring with the (DACH)Pt fragment. The malonato-Tam and malonato-hydroxy-Tam compounds were prepared through McMurry coupling of the appropriate ketones. The presence of the malonate group resulted in a pronounced stereospecificity in the reaction, since malonato-Tam was obtained only as the Z isomer, while malonato-hydroxy-Tam was obtained as an 80/20 E/Z mixture. Attribution of the isomeric structures was achieved by 2D NMR spectroscopy. The platinum complexes (DACH)Pt-malonato-Tam and (DACH)Pt-malonato-hydroxy-Tam were then prepared by coupling the barium salts derived from the malonato-Tam and malonato-hydroxy-Tam with the nitrate derived from (DACH)PtCl(2). Study of the biochemical properties of these two platinum complexes showed that, while the hydroxy-Tam complex is satisfactorily recognized by the estrogen receptor (relative binding affinity, RBA=6.4 %), the Tam complex is less well recognized (RBA=0.5 %). The effects of these complexes on two hormone-dependent breast cancer cell lines (MCF7 and MVLN) were studied in vitro. Both complexes showed an antiproliferative effect on MCF7 cells, and an antiestrogenic effect on MVLN cells. The observed effects appear to be essentially antihormonal, since incorporation of the (DACH)Pt fragment into the tamoxifen skeleton did not cause an increase in the cytotoxicity of the complexes.     

Oxidative flavour deterioration of fish oil enriched milk

The oxidative deterioration of milk emulsions supplemented with 1.5 wt‐% fish oil was investigated by sensory evaluation and by determining the peroxide value and volatile oxidation products after cold storage. Two types of milk emulsions were produced, one with a highly unsaturated tuna oil (38 wt‐% of n‐3 fatty acids) and one with cod liver oil (26 wt‐% of n‐3 fatty acids). The effect of added calcium disodium ethylenediaminetetraacetate (EDTA) on oxidation was also investigated. Emulsions based on cod liver oil with a slightly elevated peroxide value (1.5 meq/kg) oxidised significantly faster than the tuna oil emulsions, having a lower initial peroxide value (0.1 meq/kg). In the tuna oil emulsions the fishy off‐flavour could not be detected throughout the storage period. Addition of 5—50 ppm EDTA significantly reduced the development of volatile oxidation products in the cod liver oil emulsions, indicating that metal chelation with EDTA could inhibit the decomposition of lipid hydroperoxides in these emulsions. This study showed that an oxidatively stable milk emulsion containing highly polyunsaturated tuna fish oil could be prepared without significant fishy off‐flavour development upon storage, provided that the initial peroxide value was sufficiently low.     

Interlaboratory study on lipid oxidation during accelerated storage trials with rapeseed and sunflower oil analyzed by conjugated dienes as primary oxidation products

Accelerated storage tests are frequently used to assess the oxidative stability of foods and related systems due to its reproducibility. Various methods and experimental conditions are used to measure lipid oxidation. Differences between laboratories make it necessary to determine the repeatability and reproducibility of oxidation tests performed under the same conditions. The objective of the present interlaboratory study was to evaluate the outcome of a storage test for two different bulk oils, sunflower oil (SFO) and rapeseed oil (RSO), during a period of 9 weeks at 20°C, 30°C, 40°C, and 60°C. Sixteen laboratories were provided with bottled oils and conducted the storage tests according to a detailed protocol. Lipid oxidation was monitored by the formation of conjugated dienes (CD) and the activation energy (E_a) was determined for comparative purposes and statistically evaluated. An increase in CD formation was observed for both oils when the storage temperature was increased in all laboratories. The E_a,1 ranged from 47.9 to 73.3 kJ mol⁻¹ in RSO and from 27.8 to 62.6 kJ mol⁻¹ in SFO, with average values of 58.2 and 46.8 kJ mol⁻¹, respectively. The reproducibility coefficients were 10.9% and 18.2% for RSO and SFO, respectively. Practical applications: In order to compare results on oxidative stability of foods derived from different studies, the reproducibility of storage tests and methods employed to evaluate the oxidation level should be considered. This study provides fundamental data on the reproducibility of lipid oxidation under accelerated storage conditions and defines important parameters to be considered for the conduction of experiments.     

Temperature-Dependent Re-alignment of the Short Multifunctional Peptide BP100 in Membranes Revealed by Solid-State NMR Spectroscopy and Molecular Dynamics Simulations

BP100 is a cationic undecamer peptide with antimicrobial and cell-penetrating activities. The orientation of this amphiphilic α-helix in lipid bilayers was examined under numerous conditions using solid-state ¹⁹F, ¹⁵N and ²H NMR. At high temperatures in saturated phosphatidylcholine lipids, BP100 lies flat on the membrane surface, as expected. Upon lowering the temperature towards the lipid phase transition, the helix is found to flip into an upright transmembrane orientation. In thin bilayers, this inserted state was stable at low peptide concentration, but thicker membranes required higher peptide concentrations. In the presence of lysolipids, the inserted state prevailed even at high temperature. Molecular dynamics simulations suggest that BP100 monomer insertion can be stabilized by snorkeling lysine side chains. These results demonstrate that even a very short helix like BP100 can span (and thereby penetrate through) a cellular membrane under suitable conditions.     

Model-based control of titer and glycosylation in fed-batch mAb production: Modeling and control system development

Therapeutic monoclonal antibodies (mAbs) are typically manufactured using mammalian cell cultures in fed-batch bioreactors, with increasing emphasis on meeting productivity and product quality attribute targets that depend strongly on such process variables as nutrient feed rates and bioreactor operating conditions. In this article, we identify, categorize, and address the challenges of achieving both productivity and product quality goals simultaneously, by developing a multivariable, model-based control system that can satisfy multiple production objectives in a fed-batch cell culture process. Here, we discuss model development and present theoretical concepts of observability and controllability that are essential to understanding and handling effectively these intrinsic challenges. Subsequently, we evaluate via simulation the performance of the outer-loop model predictive control and demonstrate the overall capability to satisfy complex production objectives in a laboratory scale bioreactor, as a first step toward the ultimate goal of creating an advanced control system for fed-batch mAb manufacturing processes.