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.     

Role of Serum Cholesterol and Statin Use in the Risk of Prostate Cancer Detection and Tumor Aggressiveness

The aim of this study was to analyze the relationship between statin use along with serum cholesterol levels and prostate cancer (PCa) detection and aggressiveness. Statin users of three years or more and serum cholesterol levels (SC) were assessed in 2408 men scheduled for prostate biopsy. SC was classified as normal (NSC: <200 mg/dL) or high (HSC: >200 mg/dL). High-grade PCa (HGPCa) was considered if the Gleason score was greater than 7. Statin users comprised 30.9% of those studied. The PCa detection rate was 31.2% of men on statins and 37% of non-statin users (p < 0.006). The PCa detection rate was 26.3% in men with NSC and 40.6% in those with HSC (p < 0.001). In the subset of NSC men, the PCa rate was 26.5% for statin users and 26.2% for non-users (p = 0.939), while in men with HSC, the PCa rate was 36.4% for statin users and 42.0% for non-statin users (p = 0.063). The HGPCa rate was 41.8% for statin users and 32.5% for non-users (p = 0.012). NSC men had a 53.8% rate of HGPCa, while the rate was only 27.6% in HSC men (p < 0.001). NSC men on statins had an HGPCa rate of 70.2%, while non-statin users had a rate of 41.2% (p < 0.001). The HGPCa rate for HSC men on statins was 18.8%, while the rate was 30.0% (p = 0.011) for non-users. Logistic regression analysis suggested that serum cholesterol levels could serve as an independent predictor of PCa risk, OR 1.87 (95% CI 1.56–2.24) and HGPCa risk, OR 0.31 (95% CI 0.23–0.44), while statin usage could not. Statin treatment may prevent PCa detection through serum cholesterol-mediated mechanisms. A disturbing increase in the HGPCa rate was observed in statin users who normalized their serum cholesterol.     

Taioba (Xanthosoma sagittifolium) Leaves: Nutrient Composition and Physiological Effects on Healthy Rats

Several studies have shown that fruits and vegetables contribute to protect against degenerative pathologies such as cardiovascular diseases, diabetes, and cancer, mainly due to the presence of dietary fiber (DF) and polyphenols. Taioba (Xanthosoma sagittifolium) is an edible aroid widely grown in many parts of Africa, America, and Asia. The tubers portions of taioba are widely consumed; however, the leafy portions are generally discarded, despite their high nutritive value. In this study, we have partly characterized the DF of lyophiized taioba leaf (LTL), and assessed the possible protective effects on biochemical parameters and on bile acid (BA) production in colon and cecum, when fed to healthy rats for 4 wk. Forty‐five Wistar rats were assigned to either of 5 groups: group 1 received AIN 93G diet (CG: Control); group 2 received AIN 93G containing 2.5% of cellulose + 2.5% inulin (CEIN_5%); group 3 received AIN 93G containing 2.5% of cellulose + 2.5% taioba fiber (CETA_5%); group 4 received AIN 93G containing 5% cellulose + 2.5% taioba fiber (CETA_7.5%); group 5 received AIN 93G containing 5% cellulose + 2.5% of inulin (CEIN_7.5%). LTL showed high contents of total fiber, predominantly comprising insoluble DF with glucose as the major monomer. Rats receiving LTL had increased fecal mass and fat excretion, and improved BA profiles by diminishing the proportion of secondary acids, thus suggesting that consumption of taioba leaf may have the property of lowering the risk of colon cancer.     

Amperometric Cholesterol Biosensor Using Layer-by-Layer Adsorption Technique on Polyaniline-coated Polyester Films

An amperometric cholesterol biosensor was fabricated using polyaniline-coated polyester films. Polyaniline was dissolved in chloroform with camphorsulfonic acid, and polystyrene was added to this solution. Using this mixed solution, the coating was placed onto polyester films. Cholesterol oxidase was immobilized onto these films using an electrostatic layer-by-layer adsorption technique. Poly(diallyldimethylammonium chloride) was used as the counter ion source. The level of adsorption was examined and evidence of layer-by-layer adsorption was investigated using a quartz crystal microbalance (QCM). A cholesterol biosensor was fabricated from these films as a working electrode, and it was used to measure the cholesterol concentration.     

Functional properties of cholesterol-removed whipping cream treated by beta-cyclodextrin

The present study was carried out to examine the changes in functional properties of cholesterol-removed whipping cream by beta-cyclodextrin (beta-CD) treatment. The cholesterol removal rate reached over 90% in cream before whipping in all conditions (different stirring time and speed) applied. The apparent viscosity of beta-CD treated cream after whipping increased with increased stirring time and speed. Comparatively, the overrun percentage reached to 150%, and foam instability was measured as 2.5 ml deformed cream with lower stirring time (10 min) and speed (400 rpm). The thiobarbituric acid value of cholesterol-removed whipping cream increased from 0.08 to 0.14 stored at 4 degrees C during 4 wk; however, no difference was found compared with that of control. Above results indicated that beta-CD treatment process for cholesterol removal did not show a profound adverse effect on functional properties of cream after whipping.     

Dietary cholesterol impairs memory and memory increases brain cholesterol and sulfatide levels.

Cholesterol and sulfatides play many important roles in learning and memory. To date, our observations about the effects of cholesterol on learning have been assessed during response acquisition; that is, the learning of a new memory. Here, we report for the first time to our knowledge, on the effect of a cholesterol diet on a previously formed memory. Rabbits were given trace conditioning of the nictitating membrane response for 10 days, then fed a 2% cholesterol diet for 8 weeks, and then assessed for memory recall of the initially learned task. We show that dietary cholesterol had an adverse effect on memory recall. Second, we investigated whether dietary cholesterol caused an increase in brain cholesterol and sulfatide levels in four major brain structures (hippocampus, frontal lobe, brainstem, and cerebellum) using a technique for analyzing myelin and myelin-free fractions separately. Although our data confirm previous findings that dietary cholesterol does not directly affect cholesterol and establish that it does not affect sulfatide levels in the brain, these levels did increase rather significantly in the hippocampus and frontal lobe as a function of learning and memory. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Antifriction plastic greases based on intermediate products of oil refining and liquid crystalline compounds

The rheological and triboengineering properties are presented of hydrated calcium plastic greases based on intermediate products of oil refining (hydrofined vacuum gas oil and vacuum distillate). It is shown that application of liquid crystalline cholesterol compounds as an additional component of the dispersion medium of plastic greases permits significant reduction of the wear and losses for friction and boosts the load—bearing capacity of tribocouples.     

Advances in the Study of the Antiatherogenic Function and Novel Therapies for HDL

The hypothesis that raising high-density lipoprotein cholesterol (HDL-C) levels could improve the risk for cardiovascular disease (CVD) is facing challenges. There is multitudinous clear clinical evidence that the latest failures of HDL-C-raising drugs show no clear association with risks for CVD. At the genetic level, recent research indicates that steady-state HDL-C concentrations may provide limited information regarding the potential antiatherogenic functions of HDL. It is evident that the newer strategies may replace therapeutic approaches to simply raise plasma HDL-C levels. There is an urgent need to identify an efficient biomarker that accurately predicts the increased risk of atherosclerosis (AS) in patients and that may be used for exploring newer therapeutic targets. Studies from recent decades show that the composition, structure and function of circulating HDL are closely associated with high cardiovascular risk. A vast amount of data demonstrates that the most important mechanism through which HDL antagonizes AS involves the reverse cholesterol transport (RCT) process. Clinical trials of drugs that specifically target HDL have so far proven disappointing, so it is necessary to carry out review on the HDL therapeutics.     

Fibroblast Growth Factor 21 Response in a Preclinical Alcohol Model of Acute-on-Chronic Liver Injury

Background and Aims: Fibroblast growth factor (FGF) 21 has recently been shown to play a potential role in bile acid metabolism. We aimed to investigate the FGF21 response in an ethanol-induced acute-on-chronic liver injury (ACLI) model in Abcb4^−/− mice with deficiency of the hepatobiliary phospholipid transporter. Methods: Total RNA was extracted from wild-type (WT, C57BL/6J) and Abcb4⁻/⁻ (KO) mice, which were either fed a control diet (WT-Cont and KO-Cont groups; n = 28/group) or ethanol diet, followed by an acute ethanol binge (WT-EtOH and KO-EtOH groups; n = 28/group). A total of 58 human subjects were recruited into the study, including patients with alcohol-associated liver disease (AALD; n = 31) and healthy controls (n = 27). The hepatic and ileal expressions of genes involved in bile acid metabolism, plasma FGF levels, and bile acid and its precursors 7α- and 27-hydroxycholesterol (7α- and 27-OHC) concentrations were determined. Primary mouse hepatocytes were isolated for cell culture experiments. Results: Alcohol feeding significantly induced plasma FGF21 and decreased hepatic Cyp7a1 levels. Hepatic expression levels of Fibroblast growth factor receptor 1 (Fgfr1), Fgfr4, Farnesoid X-activated receptor (Fxr), and Small heterodimer partner (Shp) and plasma FGF15/FGF19 levels did not differ with alcohol challenge. Exogenous FGF21 treatment suppressed Cyp7a1 in a dose-dependent manner in vitro. AALD patients showed markedly higher FGF21 and lower 7α-OHC plasma levels while FGF19 did not differ. Conclusions: The simultaneous upregulation of FGF21 and downregulation of Cyp7a1 expressions upon chronic plus binge alcohol feeding together with the invariant plasma FGF15 and hepatic Shp and Fxr levels suggest the presence of a direct regulatory mechanism of FGF21 on bile acid homeostasis through inhibition of CYP7A1 by an FGF15-independent pathway in this ACLI model. Lay Summary: Alcohol challenge results in the upregulation of FGF21 and repression of Cyp7a1 expressions while circulating FGF15 and hepatic Shp and Fxr levels remain constant both in healthy and pre-injured livers, suggesting the presence of an alternative FGF15-independent regulatory mechanism of FGF21 on bile acid homeostasis through the inhibition of Cyp7a1.