Isolation and characterization of a Δ-9 fatty acid desaturase gene from the oleaginous yeast Cryptococcus curvatus CBS 570

The oleaginous yeast Cryptococcus curvatus is of industrial interest because it can accumulate triacylglycerols up to 60% of the cell dry weight. We are aiming at genetic modification of fatty acid biosynthesis for the production of tailor-made triacylglycerols in C. curvatus. As a first step in the development of a transformation and expression system a gene encoding the Δ-9 fatty acid desaturase of C. curvatus (CBS 570) was cloned. The 1470 bp gene encodes a protein of 493 amino acids with a calculated molecular mass of 55 kDa. The gene shows strong similarity to previous cloned Δ-9 desaturase genes from rat and Saccharomyces cerevisiae, 62 and 72%, respectively. Expression of the Δ-9 desaturase gene was studied. Supplementation of the growth medium with oleic acid (C18:1(c9)) showed a strong repression (90%) on the mRNA level, while supplementation with petroselinic acid (C18:1(c6)) had no effect on the amount of mRNA.     

Adipose Stromal/Stem Cell-Derived Extracellular Vesicles: Potential Next-Generation Anti-Obesity Agents

Over the last decade, several compounds have been identified for the treatment of obesity. However, due to the complexity of the disease, many pharmacological interventions have raised concerns about their efficacy and safety. Therefore, it is important to discover new factors involved in the induction/progression of obesity. Adipose stromal/stem cells (ASCs), which are mostly isolated from subcutaneous adipose tissue, are the primary cells contributing to the expansion of fat mass. Like other cells, ASCs release nanoparticles known as extracellular vesicles (EVs), which are being actively studied for their potential applications in a variety of diseases. Here, we focused on the importance of the contribution of ASC-derived EVs in the regulation of metabolic processes. In addition, we outlined the advantages/disadvantages of the use of EVs as potential next-generation anti-obesity agents.     

Assembly and Characterization of Biocompatible Coenzyme Q10-Enriched Lipid Nanoparticles

Coenzyme Q₁₀ (CoQ₁₀) is a strongly hydrophobic lipid that functions in the electron transport chain and as an antioxidant. CoQ₁₀ was conferred with aqueous solubility by incorporation into nanoparticles containing phosphatidylcholine (PtdCho) and apolipoprotein (apo) A-I. These particles, termed CoQ₁₀ nanodisks (ND), contain 1.0 mg CoQ₁₀/5 mg PtdCho/2 mg apoA-I (97% CoQ₁₀ solubilization efficiency). UV/Vis absorbance spectroscopy of CoQ₁₀ ND revealed a characteristic absorbance peak centered at 275 nm. Incorporation of CoQ₁₀ into ND resulted in quenching of apoA-I tryptophan fluorescence emission. Gel filtration chromatography of CoQ₁₀ ND gave rise to a single major absorbance peak and HPLC of material extracted from this peak confirmed the presence of CoQ₁₀. Incubation of cultured cells with CoQ₁₀ ND, but not empty ND, resulted in a significant increase in the CoQ₁₀ content of mitochondria as well as enhanced oxidative phosphorylation, as observed by a ~24% increase in maximal oxygen consumption rate. Collectively, a facile method to solubilize significant quantities of CoQ₁₀ in lipid nanoparticles has been developed. The availability of CoQ₁₀ ND provides a novel means to investigate biochemical aspects of CoQ₁₀ uptake by cells and/or administer it to subjects deficient in this key lipid as a result of inborn errors of metabolism, statin therapy, or otherwise.     

A multiplex RTi-PCR reaction for simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Staphylococcus aureus on fresh, minimally processed vegetables

In this work, a new multiplex single-tube real-time PCR approach is presented for the detection of Escherichia coli O157:H7, Salmonella spp. and Staphylococcus aureus, three of the more frequent food-borne bacterial pathogens that are usually investigated in a variety of food matrices. The study includes the design and specificity testing, of a new primer and probe specific for Salmonella spp. Reaction conditions were adjusted for the simultaneous amplification and detection of specific fragments in the beta-glucuronidase (uidA, E. coli) and Thermonulease (nuc, Sta. aureus) genes, and in the replication origin sequence (oriC, Salmonella spp.). Melting-curve analysis using a SYBR Green I RTi-PCR approach showed characteristic T(m) values demonstrating the specific and efficient amplification of the three fragments. Subsequently, a TaqMan RTi-PCR approach was settled, using FAM, NED and VIC fluorescently labelled specific probes for an automated detection. It was equally sensitive than uniplex RTi-PCR reactions in Sta. aureus and E. coli O157:H7, using same amounts of purified DNA, and allowed detection of 10 genome equivalents in the presence of 10(2) or 10(4) genome equivalents of the other two pathogens. Finally, it was tested in artificially inoculated fresh, minimally processed vegetables, revealing a sensitivity of 10(3)CFUg(-1) each of these pathogens in direct detection, following DNA extraction with DNeasy Tissue Kit (Qiagen). The multiplex RTi-PCR developed scored the sensitivity recognised for PCR in food and it allows a high-throughput and automation, thus it is promising as a rapid and cost-effective test for the food industry.     

A DNA Methyltransferase Modulator Inspired by Peyssonenyne Natural Product Structures

A novel epigenetic modulator that displays a DNMT1 inhibition and DNMT3A activation profile was characterized (compound 8 ). This compound is a derivative of palmitic acid that incorporates the putative reactive functional group (diynone) of the peyssonenyne natural products. Other analogues containing the diynone or an acetoxyenediyne did not show the same biological profile. In U937 human leukemia cells, diynone 8 induced cell differentiation and apoptosis, which correlated with the expression of Fas protein. Very surprisingly, diynone 8 was toxic to normal human fibroblasts (BJ) and mouse embryo fibroblasts (MEF), but not to immortalized human fibroblasts (BJEL); this unique effect was not observed with the classical DNMT inhibitor 5‐azacytidine. Therefore, compound 8 interferes in a very specific manner with signaling pathways, the activities of which differ between normal and immortalized cell types. This toxicity is reminiscent of the effects of Dnmt1 ablation on mouse fibroblasts. In fact, some of the genes deregulated by the loss of Dnmt1 are similarly deregulated by 8 , but not by the DNMT inhibitor SGI‐1027.     

Local Notions of Healthy Eating and National Dietary Guidelines: A Comparison in Vulnerable Salvadoran Communities

This study presents an assessment of local definitions and perceptions concerning healthy eating through a study in four resource-poor border communities in El Salvador. The study included focus groups, key-informant interviews, and observations of the food environment. Local definitions of healthy eating elicited through focus groups were compared to the national Salvadoran dietary guidelines recommendations. The comparison revealed several areas of overlap (including the importance of dietary variety, fruits, and vegetables, among others) and omissions (mention of limiting sweets/candy, salt, sugar, and alcohol). Focus group participants expressed concerns over the origin of their foods and whether food contained harmful chemicals. These conversations also revealed the contradictions between nutrition knowledge and preferences for foods classified as unhealthy. This article concludes with a discussion about barriers to healthy eating identified in the focus groups and through the food environment assessment.     

重组华根霉脂肪酶协同转谷氨酰胺酶提高冷冻面团抗冻发酵特性的研究

采用差示扫描量热仪和F3流变发酵仪研究重组华根霉脂肪酶(RCL)和转谷氨酰胺酶(TG)共同作用对冷冻面团抗冻发酵特性的影响。将面团于-18℃冻藏0、7、21、35d,结果发现:随着冻藏时间的延长,甘油含量有所降低,可冻结水含量增加;引入RCL和TG到冷冻面团中可以显著增加面团中的甘油含量,显著降低面团中可冻结水的含量,减少冰晶体的形成,并且可以提高酵母的存活数。F3流变发酵仪测定面团的发酵流变学特性,结果表明:RCL和TG同时作用可以显著降低冻藏对面团发酵高度的削弱作用,改善酵母的发酵性能和增加面团的持气率。     

Genome-wide bacterial toxicity screening uncovers the mechanisms of toxicity of a cationic polystyrene nanomaterial

By exploiting a genome-wide collection of bacterial single-gene deletion mutants, we have studied the toxicological pathways of a 60-nm cationic (amino-functionalized) polystyrene nanomaterial (PS-NH(2)) in bacterial cells. The IC(50) of commercially available 60 nm PS-NH(2) was determined to be 158 μg/mL, the IC(5) is 108 μg/mL, and the IC(90) is 190 μg/mL for the parent E. coli strain of the gene deletion library. Over 4000 single nonessential gene deletion mutants of Escherichia coli were screened for the growth phenotype of each strain in the presence and absence of PS-NH(2). This revealed that genes clusters in the lipopolysaccharide biosynthetic pathway, outer membrane transport channels, ubiquinone biosynthetic pathways, flagellar movement, and DNA repair systems are all important to how this organism responds to cationic nanomaterials. These results, coupled with those from confirmatory assays described herein, suggest that the primary mechanisms of toxicity of the 60-nm PS-NH(2) nanomaterial in E. coli are destabilization of the outer membrane and production of reactive oxygen species. The methodology reported herein should prove generally useful for identifying pathways that are involved in how cells respond to a broad range of nanomaterials and for determining the mechanisms of cellular toxicity of different types of nanomaterials.     

In vitro incorporation of14C-acetate into lipids of hamster flank organ (Costovertebral organ) sebaceous glands and epidermis

Following in vitro incubation of flank organs from male golden Syrian hamsters with sodium [1-¹⁴C] acetate, sebaceous glands and appendage-freed epidermis were obtained by treating the flank organ tissue with calcium chloride. This method permitted the study of incorporation of carbon-14 into the lipids of these skin components. Extracted lipids were identified by thin layer chromatography and autoradiography and were quantitated by liquid scintillation counting. Mono-, di-, and triglycerides, free sterols, fatty acids, wax monoesters, and squalene were identified as products of sebaceous gland metabolism of labeled acetate. In marked contrast, little incorporation of¹⁴C into triglycerides by the epidermal preparations was noted, although the epidermal lipids showed higher relative proportions of free sterols and polar lipids (including phospholipids). Accumulation of sterol esters did not occur. In both preparations phosphatidylcholine represented the major labeled phospholipid.