Lipidomic Profile of Rhodotorula toruloides by GC/MS and Antioxidant Capacity of the Oil by DPPH and TLC-Plate Methods

This work was undertaken to evaluate the antioxidant capacity of Rhodotorula toruloides lipid extract in TLC plate, using the (DPPH) (1,1-diphenyl-2-picril-  hydrazine) method as an innovative way to visualise lipid groups that comprise this activity. Similarly, carotenoids and crude oil were analysed for  antioxidant capacity by the DPPH and β-carotene/linoleic acid methods. The lipidomic profile extract analysis was performed by GC/MS and HPLC/DAD.  The sample preparation for the GC/MS analysis was made by ultrasound-assisted transesterification. Free compounds were silylated with BSTFA (N,O-Bis  (trimethylsilyl) trifluoracetamide) + 1% TMCS (Trimethylchlorosilane). The analysis of the lipid extract showed that in the saponifiable fraction saturated  fatty acids (SFA) and monounsaturated fatty acids (MUFA) were present; and in the unsaponifiable fraction were steroids and carotenoids. The antioxidant  capacity was expressed as IC50 reaching 6.4 mg/L that means relative efficiency. The oil profile, using TLC, shows the chemical groups:  carotenoids, acylglycerols, free fatty acids and steroids. Similarly, the GC/ MS analysis shows the fatty acids and steroids. The HPLC analysis describes the  carotenoids profile, highlighting b-carotene as the majority and the presence of β-carotene-5,8-epoxide, zeaxanthin and b-cryptoxanthin, characterising  the lipidomic study of this yeast

Lessons learned from the London Exercise and Pregnant (LEAP) Smokers randomised controlled trial process evaluation : implications for the design of physical activity for smoking cessation interventions during pregnancy

BACKGROUND: The challenges of delivering interventions for pregnant smokers have been poorly documented. Also, the process of promoting a physical activity intervention for pregnant smokers has not been previously recorded. This study describes the experiences of researchers conducting a randomised controlled trial of physical activity as an aid to smoking cessation during pregnancy and explores how the effectiveness of future interventions could be improved. METHODS: Two focus groups, with independent facilitators, were conducted with six researchers who had enrolled pregnant smokers in the LEAP trial, provided the interventions, and administered the research measures. Topics included recruitment, retention and how the physical activity intervention for pregnant smokers was delivered and how it was adapted when necessary to suit the women. The focus groups were audio-recorded, transcribed verbatim and subjected to thematic analysis. RESULTS: Five themes emerged related to barriers or enablers to intervention delivery: (1) nature of the intervention; (2) personal characteristics of trial participants; (3) practical issues; (4) researchers' engagement with participants; (5) training and support needs. Researchers perceived that participants may have been deterred by the intensive and generic nature of the intervention and the need to simultaneously quit smoking and increase physical activity. Women also appeared hampered by pregnancy ailments, social deprivation, and poor mental health. Researchers observed that their status as health professionals was valued by participants but it was challenging to maintain contact with participants. Training and support needs were identified for dealing with pregnant teenagers, participants' friends and family, and post-natal return to smoking. CONCLUSIONS: Future exercise interventions for smoking cessation in pregnancy may benefit by increased tailoring of the intervention to the characteristics of the women, including their psychological profile, socio-economic background, pregnancy ailments and exercise preferences. Delivering an effective physical activity intervention for smoking cessation in pregnancy may require more comprehensive training for those delivering the intervention, particularly with regard to dealing with teenage smokers and smokers' friends and family, as well as for avoiding post-natal return to smoking. TRIAL REGISTRATION: ISRCTN48600346 , date of registration: 21/07/2008

A new class of glycomimetic drugs to prevent free fatty acid-induced endothelial dysfunction

Background: Carbohydrates play a major role in cell signaling in many biological processes. We have developed a set of glycomimetic drugs that mimic the structure of carbohydrates and represent a novel source of therapeutics for endothelial dysfunction, a key initiating factor in cardiovascular complications. Purpose: Our objective was to determine the protective effects of small molecule glycomimetics against free fatty acid­induced endothelial dysfunction, focusing on nitric oxide (NO) and oxidative stress pathways. Methods: Four glycomimetics were synthesized by the stepwise transformation of 2,5­dihydroxybenzoic acid to a range of 2,5­substituted benzoic acid derivatives, incorporating the key sulfate groups to mimic the interactions of heparan sulfate. Endothelial function was assessed using acetylcholine­induced, endotheliumdependent relaxation in mouse thoracic aortic rings using wire myography. Human umbilical vein endothelial cell (HUVEC) behavior was evaluated in the presence or absence of the free fatty acid, palmitate, with or without glycomimetics (1µM). DAF­2 and H2DCF­DA assays were used to determine nitric oxide (NO) and reactive oxygen species (ROS) production, respectively. Lipid peroxidation colorimetric and antioxidant enzyme activity assays were also carried out. RT­PCR and western blotting were utilized to measure Akt, eNOS, Nrf­2, NQO­1 and HO­1 expression. Results: Ex vivo endothelium­dependent relaxation was significantly improved by the glycomimetics under palmitate­induced oxidative stress. In vitro studies showed that the glycomimetics protected HUVECs against the palmitate­induced oxidative stress and enhanced NO production. We demonstrate that the protective effects of pre­incubation with glycomimetics occurred via upregulation of Akt/eNOS signaling, activation of the Nrf2/ARE pathway, and suppression of ROS­induced lipid peroxidation. Conclusion: We have developed a novel set of small molecule glycomimetics that protect against free fatty acidinduced endothelial dysfunction and thus, represent a new category of therapeutic drugs to target endothelial damage, the first line of defense against cardiovascular disease

Kinetics Of Gas-phase Hydrolysis Of Ethyl Acetate Catalyzed By Immobilized Lipase.

Reactions catalyzed by supported enzymes present important advantages when compared with those in aqueous media or organic solvents: separation of enzymes from substrate is easily accomplished, enzyme stability may be improved, and control of the reaction products is more accurate. We present the experimental results of the kinetic study of ethyl acetate hydrolysis in gaseous phase catalyzed by a commercial immobilized lipase (Lipozyme IM; Novo Nordisk). The hydrolysis reaction was studied as a function of ethyl ester and water partial pressure at a constant temperature of 318 K. The amount of biocatalyst used was varied between 100 and 300 mg, and the reaction was studied in a flow-through glass microreactor. Under the conditions used, water was an important parameter in the gas-phase reaction. Activation energy was 24.8 kJ/mol and the overall order of reaction was one. Finally, a Bi-Bi reaction mechanism is proposed.13623-3

Adsorption of Glucagon and Insulin on an Immobilized Metal Ion Affinity Chromatography Silica Matrix

Glucagon is a hormone that increases blood glucose concentrations and is used as a pharmaceutical product mainly in the treatment of hypoglycaemia associated with diabetes. Given both its importance and high current cost, improved purification processes are in demand. By using immobilized metal ion affinity chromatography (IMAC), we conducted tests aimed at the purification of glucagon from complex mixtures. Adsorption studies of glucagon, insulin and a mixture of the two on adsorbents of silica–IDA–Me 2+ (Cu 2+ , Ni 2+ and Zn 2+ ) were carried out. Fixed-bed chromatographic experiments were performed and the adsorption affinity verified for all three metals tested. The most promising condition for glucagon and insulin separation was achieved by using Ni 2+ as the metal ligand and desorption with a pH step gradient. Two industrial insulin-processing fractions (one glucagon-rich and the other insulin-rich) were evaluated qualitatively under these conditions, resulting in an increase in glucagon purity for both fractions with a purification factor of five for the latter

Liquefied wheat bran as carbon source and inducer in high-solids submerged cultivation of Aspergillus niger for xylanase production

High-solids submerged cultivation for microbial production of industrially relevant enzymes can be facilitated by using a liquefied substrate as carbon source and inducer. Here, liquefied wheat bran (WB) was evaluated as carbon source and inducer in high-solids submerged cultivation (20%, w/w) of a filamentous fungus, A. niger, for xylanase production. A systematic study (one factor at a time) of the use of liquefied WB under different conditions of temperature, pH, enzyme loading, and reaction time was performed. This study revealed that xylanase production was favored by a substrate liquefied using the optimum pH (4.8) and temperature conditions (50 degrees C) of the commercial enzymatic preparation (2.8 FPU/g solids), while the liquefaction time was not significant above 6 h. The proposed strategy resulted in xylanase activity 5-fold higher than for a cultivation with non-liquefied substrate (control). These findings should contribute to the development of xylanase production processes using high solids loading submerged cultivation in conventional large-scale bioreactors21CAPES - Coordenação de Aperfeiçoamento de Pessoal e Nível SuperiorCNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoEMBRAPA – Empresa Brasileira de Pesquisa AgropecuáriaFAPESP – Fundação de Amparo à Pesquisa Do Estado De São Paulonão tem401182/2014-2; 131263/2014-5não tem2014/19000-3; 2016/10636-

Transgenic Soybean Seed As Protein Expression System: Aqueous Extraction Of Recombinant Beta-glucuronidase.

Soybean is one of the plant species with potential to be used as seed-based bioreactor. As part of the downstream processing (DSP) of this technology, extraction is a key step, since it defines the composition of the solution from which the recombinant product will be purified. In the present work, the characteristics of soybean seeds used as a bioreactor were evaluated from a process engineering standpoint through analysis of the influence of pH and ionic strength on the extraction of recombinant beta-glucuronidase (rGUS). Concentrations of recombinant protein and native soybean compounds were analyzed and compared with similar data from extraction studies using transgenic corn seeds as bioreactor. Efficient rGUS extraction was obtained at pH of around 5.5 with no addition of salt. Soybean seed extracts had lower levels of co-extracted native compounds, than corn seed extracts, and should be considered as a potential plant bioreactor in terms of DSP.1601157-6

Application Of Electrochemically Produced Aluminum Hydroxide Gel For Prepurification Of Recombinant Synthetic Green Fluorescent Protein Produced In Tobacco Leaves.

The use of recombinant proteins has increased greatly in recent years, as also have increased the number of techniques and materials used for their production and purification. Among the different types of bioreactors being studied, there is a general consensus among scientists that production in green plant tissues such as leaves is more feasible. However, the presence of chlorophyll and phenolic compounds in plant extracts, which can precipitate and denature the proteins besides damaging separation membranes and gels, makes this technology impracticable on a commercial scale. In the present work, the adsorption to electrochemically produced aluminum hydroxide gel was applied as a prepurification step for recombinant synthetic green fluorescent protein (sGFP), also referred to as enhanced green fluorescent protein, produced in Nicotiana benthamiana leaves. Removal efficiencies of 99.7% of chlorophyll, 88.5% of phenolic compounds, and 38.5% of native proteins from the N. benthamiana extracts were achieved without removing sGFP from the extracts. As electrochemical preparation of aluminum hydroxide gel is a cost-effective technique, its use can substantially contribute to the development of future production platforms for recombinant proteins produced in green plant tissues of pharmaceutical and industrial interest.271029-3