Evaluation of polycaprolactone − poly‐D,L‐lactide copolymer as biomaterial for breast tissue engineering

The potential of the copolymer polycaprolactone‐co‐ poly‐d ,l ‐lactic acid (PCLLA ) as a biomaterial for scaffold‐based therapy for breast tissue engineering applications was assessed. First, the synthesized PCLLA was evaluated for its processability by means of additive manufacturing (AM ). We found that the synthesized PCLLA could be fabricated into scaffolds with an overall gross morphology and porosity similar to that of polycaprolactone. The PCLLA scaffolds possessed a compressive Young's modulus (ca 46 kPa ) similar to that of native breast (0.5 ? 25 kPa ), but lacked thermal stability and underwent thermal degradation during the fabrication process. The PCLLA scaffolds underwent rapid degradation in vitro which was characterized by loss of the scaffolds' mechanical integrity and a drastic decrease in mass‐average molar mass (M _w) and number‐average molar mass (M _n) after 4 weeks of immersion in phosphate buffer solution maintained at 37 °C. The tin‐catalysed PCLLA scaffold was also found to have cytotoxic effects on cells. Although the initial mechanical properties of the PCLLA scaffolds generally showed potential for applications in breast tissue regeneration, the thermal stability of the copolymer for AM processes, biocompatibility towards cells and degradation rate is not satisfactory at this stage. Therefore, we conclude that research efforts should be geared towards fine‐tuning the copolymer synthesizing methods. © 2016 Society of Chemical Industry     

Eicosapentaenoic and docosahexaenoic acid affect mitochondrial and peroxisomal fatty acid oxidation in relation to substrate preference

Decreased triacylglycerol synthesis within hepatocytes due to decreased diacylglycerol acyltransferase (DGAT) activity has been suggested to be an important mechanism by which diets rich in fish oil lower plasma triacylglycerol levels. New findings suggest that eicosapentaenoic acid (EPA), and not docosahexaenoic acid (DHA), lowers plasma triacylglycerol by increased mitochondrial fatty acid oxidation and decreased availability of fatty acids for triacylglycerol synthesis. To contribute to the understanding of the triacylglycerol-lowering mechanism of fish oil, the different metabolic properties of EPA and DHA were studied in rat liver parenchymal cells and isolated rat liver organelles. EPA-CoA was a poorer substrate than DHA-CoA for DGAT in isolated rat liver microsomes, and in the presence of EPA, a markedly lower value for the triacyl[³H]glycerol/diacyl[³H]glycerol ratio was observed. The distribution of [1-¹⁴C]palmitic acid was shifted from incorporation into secreted glycerolipids toward oxidation in the presence of EPA (but not DHA) in rat liver parenchymal cells. [1-¹⁴C]EPA was oxidized to a much greater extent than [1-¹⁴C]DHA in rat liver parenchymal cells, isolated peroxisomes, and especially in purified mitochondria. As the oxidation of EPA was more effective and sensitive to the CPT-I inhibitor, etomoxir, when measured in a combination of both mitochondria and peroxisomes, we hypothesized that both are involved in EPA oxidation, whereas DHA mainly is oxidized in peroxisomes. In rats, EPA treatment lowered plasma triacylglycerol and increased hepatic mitochondrial fatty acid oxidation and carnitine palmitoyltransferase (CPT)-I activity in both the presence and absence of malonyl-CoA. Whereas only EPA treatment increased the mRNA levels of CPT-I, DHA treatment increased the mRNA levels of peroxisomal fatty acyl-CoA oxidase and fatty acid binding protein more effectively than EPA treatment. In conclusion, EPA and DHA affect cellular organelles in relation to their substrate preference. The present study strongly supports the hypothesis that EPA, and not DHA, lowers plasma triacylglycerol by increased mitochondrial fatty acid oxidation.     

Stabilization of a chitinase from Serratia marcescens by Gly->Ala and Xxx->Pro mutations

This paper describes attempts to increase the kinetic stabilityof chitinase B from Serratia marcescens (ChiB) by the introductionof semi-automatically designed rigidifying mutations of theGlyAla and XxxPro type. Of 15 single mutants, several displayedsignificant increases in thermal stability, whereas most mutantsshowed minor effects. All mutations with non-marginal effectson stability clustered in a limited, surface-exposed regionof the enzyme, indicating that this region is involved in apartial unfolding process that triggers irreversible thermalinactivation (aggregation). A double mutant containing two stabilizingmutations in this region (G188A, A234P) displayed a 10-foldincrease in half-life at 57°C and a 4.2°C increase inapparent T_m. These results show that entropic stabilizationworks well for ChiB and they pinpoint a region whose unfoldingmay be crucial for the kinetic stability of this enzyme. Received June 18, 2003; revised September 3, 2003; accepted September 12, 2003.     

Partial Reduction in BRCA1 Gene Dose Modulates DNA Replication Stress Level and Thereby Contributes to Sensitivity or Resistance

BRCA1 is a well-known breast cancer risk gene, involved in DNA damage repair via homologous recombination (HR) and replication fork protection. Therapy resistance was linked to loss and amplification of the BRCA1 gene causing inferior survival of breast cancer patients. Most studies have focused on the analysis of complete loss or mutations in functional domains of BRCA1. How mutations in non-functional domains contribute to resistance mechanisms remains elusive and was the focus of this study. Therefore, clones of the breast cancer cell line MCF7 with indels in BRCA1 exon 9 and 14 were generated using CRISPR/Cas9. Clones with successful introduced BRCA1 mutations were evaluated regarding their capacity to perform HR, how they handle DNA replication stress (RS), and the consequences on the sensitivity to MMC, PARP1 inhibition, and ionizing radiation. Unexpectedly, BRCA1 mutations resulted in both increased sensitivity and resistance to exogenous DNA damage, despite a reduction of HR capacity in all clones. Resistance was associated with improved DNA double-strand break repair and reduction in replication stress (RS). Lower RS was accompanied by increased activation and interaction of proteins essential for the S phase-specific DNA damage response consisting of HR proteins, FANCD2, and CHK1.     

Produktansatzbildung beim Erhitzen und Eindampfen flüssiger Lebensmittel

Formation of deposits from liquid foods on heated surfaces during heating and evaporation . In the past, deposit formation on heated surfaces in food processing has been investigated by considering the whole of the deposited material. This precluded reaction kinetic studies and it was therefore impossible to introduce preventive measures. However, ultrafiltration of milk and whey has now furnished model systems in which deposit formation by proteins and salts can be studied separately. The tendency for deposit formation of proteins is similar to the denaturation reaction of whey proteins. The heat sensitive native proteins in the liquid are deposited preferentially on surfaces already covered with reactive proteins. The deposition of milk salts is determined by the degree of supersaturation of the solution. For milk salts deposition, an activation energy was found which is typical for diffusion processes.     

The Utsira Wind-hydrogen Project——The World＇s Largest stand-alone wind-hydrogen power system

The world＇s largest standalone wind-hydrogen power system is being demonstrated at Utsira, an island off the south west coast of Norway. This isolated community of just 230 people is the testing ground for some of the most exciting developments made by Hydro and its partners - a future independent of fossil fuel.     

Development and application of new nucleic acid-based technologies for microbial community analyses in foods

Several challenges still persist in the analysis of microorganisms in foods, particularly in studies of complex communities. Nucleic acid-based methods are promising tools in addressing new questions concerning microbial communities. We have developed several new methods in the field of nucleic acid-based microbial community analyses. These methods cover both sample preparation and detection approaches. The sample preparation method involves simplified DNA purification using paramagnetic beads. As an extension of this method, the same paramagnetic beads are used for both cell separation and DNA purification. This enables full automation. The separate detection of viable and dead bacteria is a major issue in nucleic acid-based diagnostics. We have applied a living/dead dye that binds covalently to DNA and inhibits the PCR from dead cells. In addition, a DNA array-based detection assay has been developed. The assay combines the specificity obtained by enzymatic labeling of DNA probes with the possibility of detecting several targets simultaneously by DNA array hybridization. In combination with 16S rDNA amplification, this is a promising tool for community analyses. Also, we have developed a novel approach for multiplex quantitative PCR. The multiplex PCR has been combined with our DNA array-based detection method. Finally, we are now in the process of adapting a system for monitoring microbial growth and death in real-time through the tagging of bacteria with green fluorescent protein (GFP) combined with fluorescence detection using a high-resolution confocal laser scanner.     

Dietary Supplementation with 22-<Emphasis Type="Italic">S</Emphasis>-Hydroxycholesterol to Rats Reduces Body Weight Gain and the Accumulation of Liver Triacylglycerol

This study explores the pharmacokinetics of 22-S-hydroxycholesterol (22SHC) in vivo in rats. We also carried out a metabolic study to explore whether the beneficial effects observed of 22SHC on glucose and lipid metabolism in vitro could be seen in vivo in rats. In the pharmacokinetic study, rats were given 50 mg/kg of [³H]22-S-hydroxycholesterol before absorption, distribution and excretion were monitored. In the metabolic study, the effect of 22SHC (30 mg/kg/day for 3 weeks) in rats on body weight gain [chow and high-fat diet (HFD)], serum lipids triacylglycerol (TAG) content and gene expression in liver and skeletal muscle were examined. Results showed that 22SHC was well absorbed after oral administration and distributed to most organs and mainly excreted in feces. Rats receiving 22SHC gained less body weight than their controls regardless whether the animals received chow diet or HFD. Moreover, we observed that animals receiving HFD had elevated levels of serum TAG while this was not observed for animals on HFD supplemented with 22SHC. The amount of TAG in liver was reduced after 22SHC treatment in animals receiving either chow diet or HFD. Gene expression analysis revealed that two genes (carnitine palmitoyltransferase 2 and uncoupling protein 3) involved in fatty acid oxidation and energy dissipation were increased in liver. Ucp3 expression (both protein and mRNA level) was increased in skeletal muscle, but insulin-stimulated glucose uptake and TAG content were unchanged. In conclusion, 22SHC seems to be an interesting model substance in the search of treatments for disorders involving aberrations in lipid metabolism.     

Dynamic force microscopy imaging of native membranes

We employed magnetic ACmode atomic force microscopy (MACmode AFM) as a novel dynamic force microscopy method to image surfaces of biological membranes in their native environments. The lateral resolution achieved under optimized imaging conditions was in the nanometer range, even when the sample was only weakly attached to the support. Purple membranes (PM) from Halobacterium salinarum were used as a test standard for topographical imaging. The hexagonal arrangement of the bacteriorhodopsin trimers on the cytoplasmic side of PM was resolved with 1.5nm lateral accuracy, a resolution similar to images obtained in contact and tapping-mode AFM. Human rhinovirus 2 (HRV2) particles were attached to mica surfaces via nonspecific interactions. The capsid structure and 2nm sized protein loops of HRV2 were routinely obtained without any displacement of the virus. Globular and filamentous structures on living and fixed endothelial cells were observed with a resolution of 5-20nm. These examples show that MACmode AFM is a favorable method in studying the topography of soft and weakly attached biological samples with high resolution under physiological conditions.