The P2X7R-NLRP3 and AIM2 Inflammasome Platforms Mark the Complexity/Severity of Viral or Metabolic Liver Damage

P2X7R-NLRP3 and AIM2 inflammasomes activate caspase-1 and the release of cytokines involved in viral-related liver disease. Little is known about their role in non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steato-hepatitis (NASH). We characterized the role of inflammasomes in NAFLD, NASH, and HCV. Gene expression and subcellular localization of P2X7R/P2X4R-NLRP3 and AIM2 inflammasome components were examined in histopathological preparations of 46 patients with biopsy-proven viral and metabolic liver disease using real-time PCR and immunofluorescence. P2X7R, P2X4R, and Caspase-1 are two- to five-fold more expressed in patients with NAFLD/NASH associated with chronic HCV infection than those with metabolic damage only (p ≤ 0.01 for all comparisons). The AIM2 inflammasome is 4.4 times more expressed in patients with chronic HCV infection, regardless of coexistent metabolic abnormalities (p = 0.0006). IL-2, a cytokine playing a pivotal role during chronic HCV infection, showed a similar expression in HCV and NASH patients (p = 0.77) but was virtually absent in NAFLD. The P2X7R-NLRP3 complex prevailed in infiltrating macrophages, while AIM2 was localized in Kupffer cells. Caspase-1 expression correlated with elastography-based liver fibrosis (r = 0.35, p = 0.02), whereas P2X7R, P2X4R, NRLP3, Caspase-1, and IL-2 expression correlated with circulating markers of disease severity. P2X7R and P2X4R play a major role in liver inflammation accompanying chronic HCV infection, especially when combined with metabolic damage, while AIM2 is specifically expressed in chronic viral hepatitis. We describe for the first time the hepatic expression of IL-2 in NASH, so far considered a peculiarity of HCV-related liver damage.     

Loxin Reduced the Inflammatory Response in the Liver and the Aortic Fatty Streak Formation in Mice Fed with a High-Fat Diet

Oxidized low-density lipoprotein (ox-LDL) is the most harmful form of cholesterol associated with vascular atherosclerosis and hepatic injury, mainly due to inflammatory cell infiltration and subsequent severe tissue injury. Lox-1 is the central ox-LDL receptor expressed in endothelial and immune cells, its activation regulating inflammatory cytokines and chemotactic factor secretion. Recently, a Lox-1 truncated protein isoform lacking the ox-LDL binding domain named LOXIN has been described. We have previously shown that LOXIN overexpression blocked Lox-1-mediated ox-LDL internalization in human endothelial progenitor cells in vitro. However, the functional role of LOXIN in targeting inflammation or tissue injury in vivo remains unknown. In this study, we investigate whether LOXIN modulated the expression of Lox-1 and reduced the inflammatory response in a high-fat-diet mice model. Results indicate that human LOXIN blocks Lox-1 mediated uptake of ox-LDL in H4-II-E-C3 cells. Furthermore, in vivo experiments showed that overexpression of LOXIN reduced both fatty streak lesions in the aorta and inflammation and fibrosis in the liver. These findings were associated with the down-regulation of Lox-1 in endothelial cells. Then, LOXIN prevents hepatic and aortic tissue damage in vivo associated with reduced Lox-1 expression in endothelial cells. We encourage future research to understand better the underlying molecular mechanisms and potential therapeutic use of LOXIN.     

Engineered Sleeping Beauty Transposon as Efficient System to Optimize Chimp Adenoviral Production

Sleeping Beauty (SB) is the first DNA transposon employed for efficient transposition in vertebrate cells, opening new applications for genetic engineering and gene therapies. A transposon-based gene delivery system holds the favourable features of non-viral vectors and an attractive safety profile. Here, we employed SB to engineer HEK293 cells for optimizing the production of a chimpanzee Adenovector (chAd) belonging to the Human Mastadenovirus C species. To date, chAd vectors are employed in several clinical settings for infectious diseases, last but not least COVID-19. A robust, efficient and quick viral vector production could advance the clinical application of chAd vectors. To this aim, we firstly swapped the hAd5 E1 with chAd-C E1 gene by using the CRISPR/Cas9 system. We demonstrated that in the absence of human Ad5 E1, chimp Ad-C E1 gene did not support HEK293 survival. To improve chAd-C vector production, we engineered HEK293 cells to stably express the chAd-C precursor terminal protein (ch.pTP), which plays a crucial role in chimpanzee Adenoviral DNA replication. The results indicate that exogenous ch.pTP expression significantly ameliorate the packaging and amplification of recombinant chAd-C vectors thus, the engineered HEK293ch.pTP cells could represent a superior packaging cell line for the production of these vectors.     

Fighting the Huntington’s Disease with a G-Quadruplex-Forming Aptamer Specifically Binding to Mutant Huntingtin Protein: Biophysical Characterization,In Vitro and In Vivo Studies

A set of guanine-rich aptamers able to preferentially recognize full-length huntingtin with an expanded polyglutamine tract has been recently identified, showing high efficacy in modulating the functions of the mutated protein in a variety of cell experiments. We here report a detailed biophysical characterization of the best aptamer in the series, named MS3, proved to adopt a stable, parallel G-quadruplex structure and show high nuclease resistance in serum. Confocal microscopy experiments on HeLa and SH-SY5Y cells, as models of non-neuronal and neuronal cells, respectively, showed a rapid, dose-dependent uptake of fluorescein-labelled MS3, demonstrating its effective internalization, even in the absence of transfecting agents, with no general cytotoxicity. Then, using a well-established Drosophila melanogaster model for Huntington’s disease, which expresses the mutated form of human huntingtin, a significant improvement in the motor neuronal function in flies fed with MS3 was observed, proving the in vivo efficacy of this aptamer.     

Immunochemical or fluorescent labeling of vesicular subcellular fractions for microscopy imaging

We describe a procedure for the labeling of membranous vesicular purified subcellular fractions, to image them, typically by confocal laser scanning microscopy. Being intracellular organelles, these fractions, once purified cannot be attached to glass slides as for cells. Fractions are labeled “in batch” without prior embedding or freezing. Each labeling step performed by passages of resuspension/centrifugation is followed by washings. Then samples are dispersed on the glass slides. Mammalian retinal rod outer segment disks, intact brain stem myelin vesicles, and brain synaptosomes were chosen, as these subcellular fractions can be purified by well established procedures. These fractions were immunolabeled with specific antibodies. Moreover, by the earlier procedure, we show that the mitochondrial vital membrane potential probe MitoTracker Deep Red 633 stains myelin vesicles and rod disks before fixation, consistently with our previous reports of a respiring capacity of these membranes. Therefore, the technique seems adequate to become an instrument to study the structure and the function of these and other subcellular fractions. Microsc. Res. Tech. 73:1086–1090, 2010. © 2010 Wiley‐Liss, Inc.     

Liquid-liquid extraction of protease from cold-adapted yeast Rhodotorula mucilaginosa L7 using biocompatible and biodegradable aqueous two-phase systems

This work aimed to optimize the extraction of an extracellular protease produced by the cold-adapted yeast Rhodotorula mucilaginosa L7 using aqueous two-phase systems (ATPS) comprising polyethylene glycol (PEG) and sodium citrate or sodium tartrate. First, the biocompatibility of the phase forming agents was assessed. The results obtained with PEG-2000, PEG-4000, and PEG-6000 demonstrated that even at large PEG concentrations (32 wt%) the protease maintains its activity after 3 h of reaction, whereas an increase in salt concentration provokes a gradual decrease in protease stability. Subsequently, the partitioning of the protease in both types of ATPS was assessed, evaluating the effect of temperature, molecular weight, and concentration of PEG on protease purification, using two 2³-full factorial designs. The best partitioning conditions were obtained in PEG-6000/sodium tartrate-based ATPS, at 30ºC (with a yield of 81.09 ± 0.66% and a purification factor of 2.51 ± 0.03). Thus, considering the biodegradable characteristics of the system, the PEG/sodium tartrate ATPS is a viable and economic low-resolution step in protease purification, with a strong potential for future industrial application.     

trans Fatty Acids in Colostrum,Mature Milk and Diet of Lactating Adolescents

The purpose of this study was to investigate the trans fatty acids (TFA) content and distribution in colostrum, mature milk, and diet of adolescent mothers, after TFA declaration in food labels became mandatory in Brazil. Participants were healthy adolescents (n 54, 15–19 years, 1–90 days postpartum) practicing exclusive breastfeeding. Milk samples were collected 3 days after delivery (colostrum) and in the third month postpartum (mature milk) by hand expression. The fatty acid composition of the milk samples was determined by gas chromatography. TFA intake corresponded to 1.23 % of total energy value. Total 18:2 TFA accounted for less than 0.5 % of the energy intake. The amount of total 18:1 TFA (mean ± SEM) was 1.9 % ± 0.14 in colostrum and 1.5 % ± 0.2 in mature milk. The total content of n‐3 PUFA was inversely correlated with the total content of 18:1 TFA in colostrum. Both in colostrum and in mature milk, vaccenic acid (11t‐18:1) was found to be the most abundant 18:1 trans isomer, followed by elaidic acid (9t‐18:1), whereas rumenic acid (9c,11t‐18:2 CLA) was the predominant 18:2 trans isomer. In conclusion, the levels of TFA of industrial sources found in the mother's diet and breast milk (colostrum and mature milk) showed a decrease in relation to those observed in studies conducted prior to the TFA labeling resolution in Brazil. However, the current low intake levels of n‐3 LCPUFA and DHA content in the milk of lactating adolescents may be insufficient for supporting adequate neurological development of the infants.     

Structure–property relationship of sodium deoxycholate based poly(ester ether)urethane ionomers for biomedical applications

New sodium deoxycholate based poly(ester ether)urethane ionomers were prepared for the development of biomedical materials. A structure–property relationship in the tested biomaterials was established by cross‐examination of the dynamic mechanical and dielectric properties, attenuated total reflection–Fourier transform infrared investigation, thermogravimetric analysis, and surface morphology characterization. A stronger ionic interaction and solvation capacity of the ions and a higher ionic conductivity were manifested in the case of poly(ethylene oxide)‐rich segments than for poly(propylene oxide)‐rich segments in these polyurethane ionomers. The molecular and ionic interactions of the bile‐salt moiety with different polyether cosoft segments influenced chain packing and conformation, supramolecular organization, and the resulting surface morphological microstructures of the polyurethane biomembranes. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 42921.