Application of Differential Pulse Voltammetry to Determine the Efficiency of Stripping Tocopherols from Commercial Fish Oil

There has been an increase in the use of electrochemical methods for monitoring antioxidant levels in a variety of disciplines due to the sensitivity, low detection limits, ease of use, low cost and rapid analysis time offered by these techniques. One technique that has received specific attention is differential pulse voltammetry. We describe a novel application of differential pulse voltammetry to quantitatively and qualitatively determine the efficiency of removing tocopherols from commercial fish oil via column chromatographic separation. The relative limits of detection and quantitation of differential pulse voltammetry are compared to HPLC for determining the removal of tocopherols from commercial fish oil. It was determined that differential pulse voltammetry can monitor the separation of commercially added antioxidants from the bulk sample via a decrease in antioxidant oxidation currents. Furthermore, the limits of detection and quantitation were found to be comparable with values obtained using HPLC for tocopherol identification and quantitation.     

Extreme thermal behaviors of polytetrafluoroethylene and random tetrafluoroethylene fluorinated copolymers

By differential scanning calorimetry (DSC), the thermal behavior of polytetrafluoroethylene (PTFE) and random fluorinated copolymers of tetrafluoroethylene-containing hexafluoropropylene (FEP copolymers) or perfluoroalkylvinylether (PFA copolymers) as comonomers was investigated. Rapid-melt crystallization was employed to provide new data about the problem of inclusion/exclusion of co-units from the homopolymer crystal lattice. Equilibrium melting points were determined and tested in light of random copolymer predictions. Both nonequilibrium and equilibrium behaviors seem to point to the inclusion of  CF₃ side groups and the exclusion of larger ones. Finally, a new value of the equilibrium melting point of PTFE is given, in good agreement with those present in the literature. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 73: 919–925, 1999     

Plastid Transformation: New Challenges in the Circular Economy Era

In a circular economy era the transition towards renewable and sustainable materials is very urgent. The development of bio-based solutions, that can ensure technological circularity in many priority areas (e.g., agriculture, biotechnology, ecology, green industry, etc.), is very strategic. The agricultural and fishing industry wastes represent important feedstocks that require the development of sustainable and environmentally-friendly industrial processes to produce and recover biofuels, chemicals and bioactive molecules. In this context, the replacement, in industrial processes, of chemicals with enzyme-based catalysts assures great benefits to humans and the environment. In this review, we describe the potentiality of the plastid transformation technology as a sustainable and cheap platform for the production of recombinant industrial enzymes, summarize the current knowledge on the technology, and display examples of cellulolytic enzymes already produced. Further, we illustrate several types of bacterial auxiliary and chitinases/chitin deacetylases enzymes with high biotechnological value that could be manufactured by plastid transformation.     

A Novel PSMA-Targeted Probe for NIRF-Guided Surgery and Photodynamic Therapy: Synthesis and Preclinical Validation

A total of 20% to 50% of prostate cancer (PCa) patients leave the surgery room with positive tumour margins. The intraoperative combination of fluorescence guided surgery (FGS) and photodynamic therapy (PDT) may be very helpful for improving tumour margin delineation and cancer therapy. PSMA is a transmembrane protein overexpressed in 90–100% of PCa cells. The goal of this work is the development of a PSMA-targeted Near InfraRed Fluorescent probe to offer the surgeon a valuable intraoperative tool for allowing a complete tumour removal, implemented with the possibility of using PDT to kill the eventual not resected cancer cells. PSMA-617 binding motif was conjugated to IRDye700DX-NHS and the conjugation did not affect the photophysical characteristics of the fluorophore. The affinity of IRDye700DX-PSMA-617 towards PCa cells followed the order of their PSMA expression, i.e., PC3-PIP > LNCaP > PC3, PC3-FLU. NIRF imaging showed a significant PC3-PIP tumour uptake after the injection of 1 or 5 nmol with a maximum tumour-to-muscle ratio (ca. 60) observed for both doses 24 h post-injection. Importantly, urine, healthy prostate, and the bladder were not fluorescent at 24 h post-injection. Flow cytometry and confocal images highlighted a co-localization of PSMA+ cells with IRDye700DX-PSMA uptake. Very interestingly, ex vivo analysis on a tumour specimen highlighted a significant PSMA expression by tumour-associated macrophages, likely attributable to extracellular vesicles secreted by the PSMA(+) tumour cells. FGS proved that IRDye700DX-PSMA was able to easily delineate tumour margins. PDT experiments showed a concentration-dependent decrease in cell viability (from 75% at 10 nM to 12% at 500 nM), whereas controls did not show any cytotoxicity. PC3-PIP tumour-bearing mice subjected to photodynamic therapy showed a delayed tumour growth. In conclusion, a novel PSMA-targeted NIRF dye with dual imaging-PDT capabilities was synthesized and displayed superior specificity compared to other small PSMA targeted molecules.     

The Polyunsaturated Fatty Acid EPA,but Not DHA,Enhances Neurotrophic Factor Expression through Epigenetic Mechanisms and Protects against Parkinsonian Neuronal Cell Death

ω-3 Polyunsaturated fatty acids (PUFAs) have been found to exert many actions, including neuroprotective effects. In this regard, the exact molecular mechanisms are not well understood. Parkinson’s disease (PD) is the second most common age-related neurodegenerative disease. Emerging evidence supports the hypothesis that PD is the result of complex interactions between genetic abnormalities, environmental toxins, mitochondrial dysfunction, and other cellular processes, such as DNA methylation. In this context, BDNF (brain-derived neurotrophic factor) and GDNF (glial cell line-derived neurotrophic factor) have a pivotal role because they are both involved in neuron differentiation, survival, and synaptogenesis. In this study, we aimed to elucidate the potential role of two PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and their effects on BDNF and GDNF expression in the SH-SY5Y cell line. Cell viability was determined using the MTT assay, and flow cytometry analysis was used to verify the level of apoptosis. Transmission electron microscopy was performed to observe the cell ultrastructure and mitochondria morphology. BDNF and GDNF protein levels and mRNA were assayed by Western blotting and RT-PCR, respectively. Finally, methylated and hydroxymethylated DNA immunoprecipitation were performed in the BDNF and GDNF promoter regions. EPA, but not DHA, is able (i) to reduce the neurotoxic effect of neurotoxin 6-hydroxydopamine (6-OHDA) in vitro, (ii) to re-establish mitochondrial function, and (iii) to increase BNDF and GDNF expression via epigenetic mechanisms.     

Tobacco Plastid Transformation as Production Platform of Lytic Polysaccharide MonoOxygenase Auxiliary Enzymes

Plant biomass is the most abundant renewable resource in nature. In a circular economy perspective, the implementation of its bioconversion into fermentable sugars is of great relevance. Lytic Polysaccharide MonoOxygenases (LPMOs) are accessory enzymes able to break recalcitrant polysaccharides, boosting biomass conversion and subsequently reducing costs. Among them, auxiliary activity of family 9 (AA9) acts on cellulose in synergism with traditional cellulolytic enzymes. Here, we report for the first time, the production of the AA9 LPMOs from the mesophilic Trichoderma reesei (TrAA9B) and the thermophilic Thermoascus aurantiacus (TaAA9B) microorganisms in tobacco by plastid transformation with the aim to test this technology as cheap and sustainable manufacture platform. In order to optimize recombinant protein accumulation, two different N-terminal regulatory sequences were used: 5′ untranslated region (5′-UTR) from T7g10 gene (DC41 and DC51 plants), and 5′ translation control region (5′-TCR), containing the 5′-UTR and the first 14 amino acids (Downstream Box, DB) of the plastid atpB gene (DC40 and DC50 plants). Protein yields ranged between 0.5 and 5% of total soluble proteins (TSP). The phenotype was unaltered in all transplastomic plants, except for the DC50 line accumulating AA9 LPMO at the highest level, that showed retarded growth and a mild pale green phenotype. Oxidase activity was spectrophotometrically assayed and resulted higher for the recombinant proteins without the N-terminal fusion (DC41 and DC51), with a 3.9- and 3.4-fold increase compared to the fused proteins.     

Ablation of Acid Ceramidase Impairs Autophagy and Mitochondria Activity in Melanoma Cells

Cutaneous melanoma is often resistant to therapy due to its high plasticity, as well as its ability to metabolise chemotherapeutic drugs. Sphingolipid signalling plays a pivotal role in its progression and metastasis. One of the ways melanoma alters sphingolipid rheostat is via over-expression of lysosomal acid ceramidase (AC), which catalyses the hydrolysis of pro-apoptotic long-chain ceramides into sphingosine and fatty acid. In this report, we examine the role of acid ceramidase in maintaining cellular homeostasis through the regulation of autophagy and mitochondrial activity in melanoma cell lines. We show that under baseline conditions, wild-type melanoma cells had 3-fold higher levels of the autophagy marker, microtubule-associated proteins 1A/1B light chain 3B (LC3 II), compared to AC-null cells. This difference was further magnified after cell starvation. Moreover, we noticed autophagy impairment in A375 AC-null cells, possibly due to local accumulation of non-metabolized ceramides. Nonetheless, we observed that AC-null cells exhibited a significant increase in mitochondrial membrane potential compared to control cells. Consistent with this observation, we found that, after total starvation, ~30% of AC-null cells undergo apoptosis compared to ~6% of wild-type cells. As expected, AC transfection restored viability in A375 AC-null cells. Together, these findings suggest that AC-null melanoma cells change and adapt their metabolism to survive in the absence of AC, although in a way that does not allow them to cope with the stress of nutrient deprivation.     

The Diversity Quality Cycle: driving culture change through innovative governance

Corporate diversity initiatives have neither yielded higher financial returns for companies nor created significantly greater equity and equality of outcome for socially disadvantaged groups within organisations. There has been a systematic failure of diversity initiatives, as the strategic business importance of diversity has been avoided. Researchers argue that effective diversity management is dependent upon appropriate structures and systems, not upon human resource management training alone. This article discusses the impact of the design, introduction and application of the ‘Diversity Quality Cycle’. This model allows for the embedding of the values of equality of opportunity and cultural diversity throughout core business functions, and for positive long-term change. A mixed-method approach was taken; Participative Action Research is the main methodology employed, within which quantitative data were generated, analysed and interpreted. The main case study organisation is a Further Education College in the West of England.     

Nanocomposites of syndiotactic polypropylene: Phase behavior and morphology

Through differential scanning calorimetry, the crystallization kinetics of syndiotactic polypropylene (sPP) nanocomposites with organically modified layered silicates has been investigated in both nonisothermal and isothermal conditions. A strong influence on the thermal behavior is exerted by the compatibilizer agent (low molecular weight isotactic polypropylene‐g‐maleic anydrhyde, iPP‐g‐MA), which increases the crystallization rate of sPP. A further strong increase in the crystallization rate is observed upon increasing the OLS content in the sample. The kinetics data have been analyzed through the Ozawa, Avrami, and Tobin methods, the first of them resulted ineffective in describing our data. Morphological analyses have been performed by polarized optical microscopy and atomic force microscopy. Spherulitic morphology is observed for neat sPP; for the sample containing only the compatibilizer the spherulites grow around the iPP‐g‐MA particles, revealing their nucleating effect on sPP crystallization; for the nanocomposites the spherulitic morphology is lost: the texture appears globular, compact; inside the globules lamellae are present, parallel one to the other. POLYM. ENG. SCI., 46:1433–1442, 2006. © 2006 Society of Plastics Engineers