Uptake of the aromatic retinoids Ro 10-9359 (etretinate) and Ro 10-1670 (acitretin), its main metabolite, delivered to cultured human fibroblasts by serum proteins. Lack of evidence for perturbation of LDL catabolism

Etretinate or acitretin are efficiently delivered to cultured human fibroblasts in the presence of low density lipoproteins, high density lipoproteins or human serum albumin. In contrast to acitretin, delivery of etretinate to fibroblasts is more efficiently achieved with human serum albumin than with lipoproteins. The uptake of etretinate and acitretin via low density lipoproteins delivery, does not take place via the low density lipoprotein-receptor endocytotic pathway but mostly through a passive exchange with the plasma membrane. However, in contrast to acitretin, the exchange of etretinate seems to occur alter binding of etretinate-loaded low density lipoproteins to the apolipoprotein B receptors. No differences are observed in binding, internalization and degradation of native, etretinate-loaded low density lipoproteins and acitretin-loaded low density lipoproteins, suggesting that the presence of these retinoids in low density lipoproteins does not alter their processing by the cells. Furthermore, the presence of these retinoids in the cells does not notably affect, under our experimental conditions, the catabolism of native low density lipoproteins.     

Comparison Study of Bone Defect Healing Effect of Raw and Processed Pyritum in Rats

To evaluate and compare the effect of raw and processed pyritum on tibial defect healing, 32 male Sprague Dawley rats were randomly divided into four groups. After tibial defect, animals were produced and grouped: sham and control group were orally administrated with distilled water (1 mL/100 g), while treatment groups were given aqueous extracts of raw and processed pyritum (1.5 g/kg) for successive 42 days. Radiographic examination showed that bone defect healing effect of the treatment groups was obviously superior compared to that of the control group. Bone mineral density of whole tibia was increased significantly after treating with pyritum. Inductively coupled plasma-optical emission spectrometry showed that the contents of Ca, P, and Mg in callus significantly increased in the treatment groups comparing with the control. Moreover, serological analysis showed that the concentration of serum phosphorus of the treatment groups significantly increased compared with that of the control group. By in vitro study, we have evaluated the effects of drug-containing serum of raw and processed pyritum on osteoblasts. It was manifested that both the drug-containing sera of raw and processed pyritum significantly increased the mRNA levels of alkaline phosphatase and collagen type I. Protein levels of phosphorylated Smad2/3 also increased. The mRNA levels of osteocalcin and transforming growth factor β (TGF-β) type I and II receptors, as well as the protein levels of TGF-β1 in the processed groups, were higher than those in the control. In summary, both raw and processed pyritum-containing sera exhibited positive effects on osteoblasts, which maybe via the TGF-β1/Smad signaling pathway. Notably, the tibia defect healing effect of pyritum was significantly enhanced after processing.     

Engineering of Thermomyces lanuginosus lipase Lip: creation of novel biocatalyst for efficient biosynthesis of chiral intermediate of Pregabalin

Efficient and highly enantioselective hydrolysis of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) is the most crucial step in chemoenzymatic synthesis of Pregabalin. By using site-saturation mutagenesis and high-throughput screening techniques, lipase Lip from Thermomyces lanuginosus DSM 10635 was engineered to improve its activity towards CNDE. The triple mutant, S88T/A99N/V116D exhibited a 60-fold improvement in specific activity for CNDE (2.35 U/mg) over the wild-type Lip (0.039 U/mg). Modeling and docking studies demonstrated that the mutant could more effectively stabilize oxygen anions in transition states and the lid of Lip in the open conformation. Additionally, the kinetic resolution of CNDE catalyzed by Escherichia coli cell overexpressing S88T/A99N/V116D mutant afforded (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid in 42.4 % conversion and 98 % ee within 20 h with a substrate loading of 1 M (255 g/l). These results demonstrated that a novel and promising biocatalyst was created for efficient chemoenzymatic manufacturing of Pregabalin.     

Alleviation of Liver Dysfunction,Oxidative Stress,and Inflammation Underlines the Protective Effects of Polysaccharides from Cordyceps cicadae on High Sugar/High Fat Diet-Induced Metabolic Syndrome in Rats

This study investigated the protective effects of two polysaccharides (CPA-1 and CPB-2) from Cordyceps cicadae against high fructose/high fat diet (HF/HFD) induced obesity and metabolic disorders in rats. Rats were either fed with normal diet or HF/HFD and treated with CPA-1 and CPB-2 (100 and 300 mg/kg) for 11 weeks. Administration of CPA-1 and CPB-2 significantly and dose dependently reduced body and liver weight, insulin and glucose tolerance, serum insulin and glucose levels. Furthermore, serum and hepatic lipid profiles, liver function enzymes and proinflammatory cytokines (TNF-α, IL-1β and IL-6) were markedly reduced. Additionally, CPA-1 and CPB-2 treatment alleviated hepatic oxidative stress by reducing lipid peroxidation level (MDA) and upregulating glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activities as well as ameliorated histological alterations through the reduction of hepatic lipid accumulation. These results suggested that the polysaccharides from C. cicadae showed protective effects against HF/HFD induced metabolic disturbances and may be considered as a dietary supplement for treating obesity.     

RGD Surface Functionalization of the Hydrophilic Acrylic Intraocular Lens Material to Control Posterior Capsular Opacification

Posterior Capsular Opacification (PCO) is the capsule fibrosis developed on implanted IntraOcular Lens (IOL) by the de-differentiation of Lens Epithelial Cells (LECs) undergoing Epithelial Mesenchymal Transition (EMT). Literature has shown that the incidence of PCO is multifactorial including the patient''s age or disease, surgical technique, and IOL design and material. Reports comparing hydrophilic and hydrophobic acrylic IOLs have shown that the former has more severe PCO. On the other hand, we have previously demonstrated that the adhesion of LECs is favored on hydrophobic compared to hydrophilic materials. By combining these two facts and contemporary knowledge in PCO development via the EMT pathway, we propose a biomimetically inspired strategy to promote LEC adhesion without de-differentiation to reduce the risk of PCO development. By surface grafting of a cell adhesion molecule (RGD peptide) onto the conventional hydrophilic acrylic IOL material, the surface-functionalized IOL can be used to reconstitute a capsule-LEC-IOL sandwich structure, which has been considered to prevent PCO formation in literature. Our results show that the innovative biomaterial improves LEC adhesion, while also exhibiting similar optical (light transmittance, optical bench) and mechanical (haptic compression force, IOL injection force) properties compared to the starting material. In addition, compared to the hydrophobic IOL material, our bioactive biomaterial exhibits similar abilities in LEC adhesion, morphology maintenance, and EMT biomarker expression, which is the crucial pathway to induce PCO. The in vitro assays suggest that this biomaterial has the potential to reduce the risk factor of PCO development.     

Plasma FA composition in familial LCAT deficiency indicates SOAT2-derived cholesteryl ester formation in humans

Mutations in the LCAT gene cause familial LCAT deficiency (Online Mendelian Inheritance in Man ID: #245900), a very rare metabolic disorder. LCAT is the only enzyme able to esterify cholesterol in plasma, whereas sterol O-acyltransferases 1 and 2 are the enzymes esterifying cellular cholesterol in cells. Despite the complete lack of LCAT activity, patients with familial LCAT deficiency exhibit circulating cholesteryl esters (CEs) in apoB-containing lipoproteins. To analyze the origin of these CEs, we investigated 24 carriers of LCAT deficiency in this observational study. We found that CE plasma levels were significantly reduced and highly variable among carriers of two mutant LCAT alleles (22.5 [4.0–37.8] mg/dl) and slightly reduced in heterozygotes (218 [153–234] mg/dl). FA distribution in CE (CEFA) was evaluated in whole plasma and VLDL in a subgroup of the enrolled subjects. We found enrichment of C16:0, C18:0, and C18:1 species and a depletion in C18:2 and C20:4 species in the plasma of carriers of two mutant LCAT alleles. No changes were observed in heterozygotes. Furthermore, plasma triglyceride-FA distribution was remarkably similar between carriers of LCAT deficiency and controls. CEFA distribution in VLDL essentially recapitulated that of plasma, being mainly enriched in C16:0 and C18:1, while depleted in C18:2 and C20:4. Finally, after fat loading, chylomicrons of carriers of two mutant LCAT alleles showed CEs containing mainly saturated FAs. This study of CEFA composition in a large cohort of carriers of LCAT deficiency shows that in the absence of LCAT-derived CEs, CEs present in apoB-containing lipoproteins are derived from hepatic and intestinal sterol O-acyltransferase 2.     

Extension of cell membrane boosting squalene production in the engineered Escherichia coli

Squalene is a lipophilic and non-volatile triterpene with many industrial applications for food, pharmaceuticals, and cosmetics. Metabolic engineering focused on optimization of the production pathway suffer from little success in improving titers because of a limited space of the cell membrane accommodating the lipophilic product. Extension of cell membrane would be a promising approach to overcome the storage limitation for successful production of squalene. In this study, Escherichia coli was engineered for squalene production by overexpression of some membrane proteins. The highest production of 612 mg/L was observed in the engineered E. coli with overexpression of Tsr, a serine chemoreceptor protein, which induced invagination of inner membrane to form multilayered structure. It was also observed an increase in unsaturated fatty acid in membrane lipids composition, suggesting cellular response to maintain membrane fluidity against squalene accumulation in the engineered strain. This study potentiates the capability of E. coli for squalene production and provides an effective strategy for the enhanced production of such compounds.