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.     

Formation of pesticide nonextractable (bound) residues in soil: magnitude, controlling factors and reversibility

The analysis of the coherent data on nonextractable (bound) residues (NER) from the literature and EU pesticide registration dossiers allows the identification of general trends, in spite of the large variability and heterogeneity of data. About 50% of the pesticides reviewed exhibit a low proportion of NER (less than 30% of the initial amount) while only 12% of pesticides have a proportion of NER exceeding 70%. The lowest proportion of NER was found for dinitroanilines (<20%), and the largest value was obtained for carbamates, and in particular dithiocarbamates. The presence of chemical reactive groups, such as aniline or phenol, tends to yield a larger proportion of NER. NER originating from N-heteroatomic ring were found to be lower than those from phenyl-ring structures. Among the environmental factors affecting the formation of NER, microbial activity has a direct and significant effect. Concerning the NER uptake or their bioavailability, consistent data suggest that only a small percentage of the total amounts of NER can be released. The analysis of NER formation kinetics showed that incubation experiments are often stopped too early to allow a correct evaluation of the NER maturation phase. Therefore, there is a need for longer term experiments to evaluate the tail of the NER formation kinetics. Still, the heterogeneity of the NER data between pesticides and for specific pesticides calls for great care in the interpretation of the data and their generalization.     

Influence of bleached palm oil on the physicochemical and sensory properties of beef patty

The effect of palm oil on the physico-chemical and sensory properties of beef liver patty were studied. Seven batches (3units per batch) of beef liver patty with different palm oil content (5, 10, 20, 30, 40 and 50%) and a control with pork fat (30%) were manufactured by component mix at 1500 rpm for 1 min and cooked in an oven at 90?°C. Physico-chemical analysis of raw and cooked samples showed improvement of emulsion stability, water binding capacity, technological yield and hardness of patties substituted with lower proportions of deodorized bleached palm oil. No significant difference (P?>?0.05) was found between physico-chemical properties of liver patty formulated with 20% palm oil and the control (formulated with 30% pork fat). Sensory attributes generated by a semi-trained panel based mainly on texture, homogeneity (colour and aspect), odour and meltiness of the patties confirmed this tendency.     

Adaptational changes in cellular fatty acid branching and unsaturation of Aeromonas species as a response to growth temperature and salinity

The effects of growth temperature and salinity on the cellular fatty acids were investigated on Aeromonas caviae, Aeromonas hydrophila and Aeromonas sobria. Under optimal growth conditions, fatty acids patterns were dominated by even-numbered chains C(16:0), C(16:1cis9), C(18:1cis11), C(12:0) and C(14:0). Growth temperature modifications induced, in the three Aeromonas species, important changes in fatty acid (i) unsaturation, (ii) branching and (iii) chain length. An important decrease in the C(18:1cis11) fatty acid content was observed for the three species below 15 degrees C and above 25 degrees C. The evolution of C(18:1cis11) and C(16:0) showed a mirror image for the three Aeromonas species. Low NaCl concentrations did not elicit significant changes in the fatty acids content of the three Aeromonas species. However, for high NaCl concentration in the medium, the growth ability was related to an important decrease of the unsaturated to saturated fatty acids ratio indicating a membrane rigidification. Thermal and salinity adaptations were branched fatty acid-dependent for A. caviae, whereas this phenomenon was less significant for A. hydrophila and A. sobria.     

Addition of protected and unprotected fish oil to diets for dairy cows. I. Effects on the yield,composition and taste of milk

Thirty Holstein cows in mid-lactation (158+/-20 DIM) were given a total mixed ration based on grass silage, maize silage and rolled barley. After a preliminary period of 1 week, this diet was supplemented with nothing (control), unprotected fish oil (3.7% of dry matter, DM), or two levels of glutaraldehyde-protected microcapsules of fish oil (1.5% and 3.0% of DM, respectively). Unprotected and protected supplements contained, respectively, 74% and 58% of DM as lipids. Cows given the unprotected supplement reduced their feed intake by > 25%. Consequently, these cows lost body weight and produced less milk. DM intake, body weight, and milk yield were unaffected by protected fish oil. Fish oil reduced both milk fat and protein percentages, and decreased the proportion of short-chain fatty acids, stearic, and oleic acids in milk fat. Milk trans C18:1 fatty acids increased in cows given both unprotected and protected fish oil. Milk fat content of very-long-chain n3 polyunsaturated fatty acids, including C20:5 and C22:6, increased with fish oil in the diet. Accordingly, the peroxide index increased and a taste panel was able to detect unusual taste in milk from cows consuming the higher level of protected fish oil and disliked the milk from cows given unprotected fish oil. In conclusion, when lactating cows consumed fish oil, milk concentration of long-chain n3 fatty acids increased and mammary de novo synthesis of fatty acids decreased, but milk yield and milk protein content were reduced, and the milk was more susceptible to oxidation and its taste was adversely affected.     

Centrifugal partition chromatography applied to the isolation of oak wood aroma precursors

Flavours extracted from oak wood during barrel ageing contribute to the organoleptic character of wines and spirits. The aim of this work was to identify the glycosidic precursors of the key volatile compounds responsible for oak wood aroma. Oak extract is a very complex matrix and, furthermore, precursors are present in very small quantities. Preparative centrifugal partition chromatography (CPC) is a promising solution for purifying the oak extract. The solvent system was selected on the basis of the partition coefficient of glycosidase enzyme activity (K_ca). Thanks to the efficacy of CPC separation, three glucoside gallates were subsequently isolated by HPLC chromatography. Vanillin-(6′-O-galloyl)-O-β-d-glucopyranoside, 3,4,5-trimethoxyphenyl-(6′-O-galloyl)-O-β-d-glucopyranoside, and (6R,9R)-3-oxo-α-ionol-9-O-(6′-O-galloyl)-β-glucopyranoside (macarangioside E) were isolated and identified. This was the first time that vanillin-(6′-O-galloyl)-O-β-d-glucopyranoside was identified and the first time that macarangioside E was isolated from oak wood. Heating macarangioside E resulted in the formation of megastigmatrienone, which has an aroma reminiscent of tobacco.     

Comparison of methods of differentiating between fresh and frozen–thawed fish or fillets

Freezing is an efficient way of storing fish. Objectively though, it is very hard to determine whether a fish has been previously frozen. Following an appraisal of various methods, we selected a physical determination (torrymeter), a physiological examination (eye lens) and three enzymatic assays (α‐glucosidase, β‐N‐acetylglucosaminidase and β‐hydroxyacyl‐CoA‐dehydrogenase) and applied them to three species: plaice (Pleuronectes platessa), whiting (Merlangus merlangus) and mackerel (Scomber scombrus). We also compared the results obtained following slow and rapid freezing and investigated how spoilage affects the torrymeter measurements and α‐glucosidase assay values. For whole fish the physical method using the torrymeter is a reliable indicator. For fish fillets we recommend the enzymatic method using the α‐glucosidase assay, which should be accompanied by measurement of the freshness to avoid confusing a frozen–thawed fish and a fish in an advanced stage of spoilage. The values noted for fresh and thawed whiting and plaice indicated cut‐off values of 0.15 for whiting and 0.5 for plaice, above which it can be asserted that the sample had been frozen. © 2002 Society of Chemical Industry     

Oregano: chemical analysis and evaluation of its antimalarial, antioxidant, and cytotoxic activities

Abstract: GC‐FID and GC‐MS analysis of essential oil from oregano leaves (Origanum compactum) resulted in the identification of 46 compounds, representing more than 98% of the total composition. Carvacrol was the predominant compound (36.46%), followed by thymol (29.74%) and p‐cymene (24.31%). Serial extractions with petroleum ether, ethyl acetate, ethanol, and water were performed on aerials parts of Origanum compactum. In these extracts, different chemical families were characterized: polyphenols (gallic acid equivalent 21.2 to 858.3 g/kg), tannins (catechin equivalent 12.4 to 510.3 g/kg), anthocyanins (cyanidin equivalent 0.38 to 5.63 mg/kg), and flavonoids (quercetin equivalent 14.5 to 54.7 g/kg). The samples (essential oil and extracts) were subjected to a screening for antioxidant (DPPH and ABTS assays) and antimalarial activities and against human breast cancer cells. The essential oil showed a higher antioxidant activity with an IC₅₀= 2 ± 0.1 mg/L. Among the extracts, the aqueous extract had the highest antioxidant activity with an IC₅₀= 4.8 ± 0.2 mg/L (DPPH assay). Concerning antimalarial activity, Origanum compactum essential oil and ethyl acetate extract showed the best results with an IC₅₀ of 34 and 33 mg/mL, respectively. In addition, ethyl acetate extract (30 mg/L) and ethanol extract (56 mg/L) showed activity against human breast cancer cells (MCF7). The oregano essential oil was considered to be nontoxic.     

Antimicrobial Activity of Nisin and Natamycin Incorporated Sodium Caseinate Extrusion‐Blown Films: A Comparative Study with Heat‐Pressed/Solution Cast Films

Antimicrobial edible films based on sodium caseinate, glycerol, and 2 food preservatives (nisin or natamycin) were prepared by classical thermomechanical processes. Food preservatives were compounded (at 65 °C for 2.5 min) with sodium caseinate in a twin‐screw extruder. Anti‐Listeria activity assays revealed a partial inactivation of nisin following compounding. Thermoplastic pellets containing food preservatives were then used to manufacture films either by blown‐film extrusion process or by heat‐press. After 24 h of incubation on agar plates, the diameters of K. rhizophila growth inhibition zones around nisin‐incorporated films prepared by solution casting (control), extrusion blowing or heat pressing at 80 °C for 7 min of nisin‐containing pellets were 15.5 ± 0.9, 9.8 ± 0.2, and 8.6 ± 1.0 mm, respectively. Since heat‐pressing for 7 min at 80 °C of nisin‐incorporated pellets did not further inactivate nisin, this indicates that nisin inactivation during extrusion‐blowing was limited. Moreover, the lower diameter of the K. rhizophila growth inhibition zone around films prepared with nisin‐containing pellets compared to that observed around films directly prepared by solution casting confirms that nisin inactivation mainly occurred during the compounding step. Natamycin‐containing thermoplastic films inhibited Aspergillus niger growth; however, by contrast with nisin‐containing films, heat‐pressed films had higher inhibition zone diameters than blown films, therefore suggesting a partial inactivation of natamycin during extrusion‐blowing.     

Inhibition of Toll-Like Receptor 4 Signaling Mitigates Microvascular Loss but Not Fibrosis in a Model of Ischemic Acute Kidney Injury

The development of chronic kidney disease (CKD) following an episode of acute kidney injury (AKI) is an increasingly recognized clinical problem. Inhibition of toll-like receptor 4 (TLR4) protects renal function in animal models of AKI and has become a viable therapeutic strategy in AKI. However, the impact of TLR4 inhibition on the chronic sequelae of AKI is unknown. Consequently, we examined the chronic effects of TLR4 inhibition in a model of ischemic AKI. Mice with a TLR4-deletion on a C57BL/6 background and wild-type (WT) background control mice (C57BL/6) were subjected to bilateral renal artery clamping for 19 min and reperfusion for up to 6 weeks. Despite the acute protective effect of TLR4 inhibition on renal function (serum creatinine 1.6 ± 0.4 mg/dL TLR4-deletion vs. 2.8 ± 0.3 mg/dL·WT) and rates of tubular apoptosis following ischemic AKI, we found no difference in neutrophil or macrophage infiltration. Furthermore, we observed significant protection from microvascular rarefaction at six weeks following injury with TLR4-deletion, but this did not alter development of fibrosis. In conclusion, we validate the acute protective effect of TLR4 signal inhibition in AKI but demonstrate that this protective effect does not mitigate the sequential fibrogenic response in this model of ischemic AKI.