Identification of Nuclear and Cytoplasmic mRNA Targets for the Shuttling Protein SF2/ASF

The serine and arginine-rich protein family (SR proteins) are highly conserved regulators of pre-mRNA splicing. SF2/ASF, a prototype member of the SR protein family, is a multifunctional RNA binding protein with roles in pre-mRNA splicing, mRNA export and mRNA translation. These observations suggest the intriguing hypothesis that SF2/ASF may couple splicing and translation of specific mRNA targets in vivo. Unfortunately the paucity of endogenous mRNA targets for SF2/ASF has hindered testing of this hypothesis. Here, we identify endogenous mRNAs directly cross-linked to SF2/ASF in different subcellular compartments. Cross-Linking Immunoprecipitation (CLIP) captures the in situ specificity of protein-RNA interaction and allows for the simultaneous identification of endogenous RNA targets as well as the locations of binding sites within the RNA transcript. Using the CLIP method we identified 326 binding sites for SF2/ASF in RNA transcripts from 180 protein coding genes. A purine-rich consensus motif was identified in binding sites located within exon sequences but not introns. Furthermore, 72 binding sites were occupied by SF2/ASF in different sub-cellular fractions suggesting that these binding sites may influence the splicing or translational control of endogenous mRNA targets. We demonstrate that ectopic expression of SF2/ASF regulates the splicing and polysome association of transcripts derived from the SFRS1, PABC1, NETO2 and ENSA genes. Taken together the data presented here indicate that SF2/ASF has the capacity to co-regulate the nuclear and cytoplasmic processing of specific mRNAs and provide further evidence that the nuclear history of an mRNA ma

Soluble starch and composite starch Bioactive Glass 45S5 particles : synthesis, bioactivity, and interaction with rat bone marrow cells

For many biomedical applications, biodegradable and simultaneously bioactive materials are desired. These materials should at the same time be able to support cell function and co-exist with the organism without triggering a relevant immune response. In this work, the synthesis as well as the bioactivity evaluation of newly developed polymer soluble potato starch and composite (with Bioactive Glass 45S5) micron-size particles are reported. An extremely interesting result is that although with different properties, both polymer and composite particles were able to form a calcium phosphate layer at their surface, which is a clear indication of their bioactivity. The cytotoxicity and the ability to support cell attachment and growth of the developed materials were also studied, and both polymer and composite materials were shown to be non-cytotoxic. Preliminary results show that both types of materials were found to allow rat bone marrow cells to attach and to proliferate on their surface and to express osteogenic markers, such as alkaline phosphatase and osteopontin. The obtained results indicate that the developed carriers might be used as substrates for cell culture in vitro, in order to form constructs that might be used as a part of a tissue engineering strategy

Fluorescence studies on new potential antitumoral 1,3-diarylurea derivatives in the thieno[3,2-b]pyridine series encapsulated in magnetoliposomes

Magnetic nanoparticles of magnetite and of nickel core with silica shell were prepared and either covered with a lipid bilayer or entrapped in liposomes, forming magnetoliposomes. New potential antitumoral 1,3-diarylurea derivatives of thieno[3,2-b]pyridines were then encapsulated in liposomes and magnetoliposomes and their photophysical behavior was investigated.Fundação para a Ciência e a Tecnologia (FCT), FEDER, COMPETE/QREN/EU for financial support to CFUM (PEst-C/FIS/UI0607/2011) and CQ/UM (PEst-C/QUI/UI0686/2011) and to research projects PTDC/QUI/81238/2006 (FCOMP-01-0124-FEDER-007467), PTDC/QUIQUI/111060/2009 (FCOMP-01-0124-FEDER-015603)

Predictors of Psychological Well-Being during Behavioral Obesity Treatment in Women

This study examined the association of autonomy-related variables, including exercise motivation, with psychological well-being and quality of life, during obesity treatment. Middle-aged overweight/obese women (n = 239) participated in a 1-year behavioral program and completed questionnaires measuring need support, general self-determination, and exercise and treatment motivation. General and obesity-specific health-related quality of life (HRQOL), self-esteem, depression, and anxiety were also assessed. Results showed positive correlations of self-determination and perceived need support with HRQOL and self-esteem, and negative associations with depression and anxiety (P < .001). Treatment autonomous motivation correlated positively with physical (P = .004) and weight-related HRQOL (P < .001), and negatively with depression (P = .025) and anxiety (P = .001). Exercise autonomous motivation was positively correlated with physical HRQOL (P < .001), mental HRQOL (P = .003), weight-related HRQOL (P < .001), and self-esteem (P = .003), and negatively with anxiety (P = .016). Findings confirm that self-determination theory's predictions apply to this population and setting, showing that self-determination, perceived need support, and autonomous self-regulation positively predict HRQOL and psychological well-being

Aqueous two-phase systems comprising ionic liquids for the extraction of recombinant proteins

The progress made on the use of new biopharmaceuticals has a high influence on the global health, and since the approval of humulin (recombinant insulin) by FDA, new protein-based pharmaceuticals have been introduced in the market such as the interferon alfa 2b (IFNα2b), which has been used in in the treatment of oncological diseases for more than 30 years. Nowadays, the (bio)pharmaceutical industry is committed to improve the quality of their products while endeavoring a reduction of its production costs. Currently, the downstream processing of biopharmaceuticals is dominated by chromatography and, despite being highly effective, is responsible for their current high-cost [1]. Accordingly, there is a crucial need to develop effective alternative purification platforms. In this way, aqueous two-phase systems (ATPS) emerge. Due to their high-water content, ATPS may be considered a biocompatible technique for the purification of (bio)molecules. Additionally, the use of ionic liquids (ILs) in low concentrations as adjuvants in ATPS affords an enhanced performance in what concerns to (bio)molecules selectivity. In this work it is shown the use of polymer-polymer ATPS comprising ILs as adjuvants to purify recombinant proteins. Several variables were studied in the extraction of the target proteins, namely pH, polymer molecular weight, and the effect of different families of ILs and their concentration. Overall, imidazolium-based ILs seem responsible for an improved ATPS performance towards a higher purity, and is additionally shown that electrostatic interactions between the negatively charged protein and ILs cations may account for this behavior.publishe

Downstream processing of recombinant proteins using alternative purification platforms based on ionic liquids

Remarkable advances in the treatment of chronic diseases have been achieved with biopharmaceutical-based therapies, a sector that is currently dominated by therapeutic proteins with over 200 protein drugs in the market1. Improvements in the biopharmaceuticals upstream stage have resulted in high yields of the desired product, and shifted the bioprocess bottleneck to the downstream processing, which is currently dominated by chromatography, accounting with more than 70% of total downstream costs2. Aiming at finding new cost-effective, efficient and sustainable technologies for proteins purification, novel polymer-polymer aqueous biphasic systems (ABS) using ionic liquids (ILs) as adjuvants were investigated in the downstream of interferon alfa 2b (IFNα2b) from Escherichia coli BL21 cultures. The phase diagrams of ABS composed by polyethylene glycol (PEG) and polypropylene glycol (PPG), with ILs as adjuvants, were determined at 25 °C. Chloride-based ILs combined with cholinium, imidazolium, pyrrolidinium, piperidinium, tetralkylammonium and tetralkylphosphonium cations were investigated. The use of ILs allows the selective removal of the protein impurities present in the culture medium. In summary, the application of ILs allowed to develop effective IFNα2b purification platforms from E. coli based on polymer-polymer-ABS, having significant benefits in terms of economic, biocompatible and environmental issues. References: 1Walsh G, Nat Biotechnol, 2018, 36, 1136-1145; 2Azevedo AM, Rosa PAJ, Ferreira IF, et al. Trends Biotechnol, 2009, 27, 240-247.publishe

Solid and aqueous magnetic liposomes as nanocarriers for a new potential drug active against breast cancer

In this work, iron oxide nanoparticles (NPs), with diameters around 12 nm, were synthesized by coprecipitation method. Both aqueous and solid magnetic liposomes containing magnetite NPs were obtained, with sizes below 140 nm. A new antitumor compound, a diarylurea derivative of thienopyridine, active against breast cancer, was incorporated in both aqueous and solid magnetoliposomes, being located mainly in the lipid membrane. A promising application of these magnetic liposomes in oncology is anticipated, allowing a combined therapeutic approach, using both chemotherapy and magnetic hyperthermia.Financial support by FCT-Portugal is acknowledged (Strategic Funding UID/FIS/04650/2013 and PhD grant SFRH/BD/90949/2012).info:eu-repo/semantics/publishedVersio

Improving the purification of recombinant proteins by the use of ionic liquids as adjuvants in aqueous two-phase systems

The development of new drugs has a high impact on the global health, and since the approval of humulin (recombinant insulin) by FDA, several protein-based biopharmaceuticals have been introduced in the market. These products are usually attained through biotechnological processes using engineered biological sources and have led to remarkable advances in the treatment of distinct human diseases, as for example the case of interferon alfa 2b (IFNα2b) which has been used in in the treatment of oncological diseases for more than 30 years [1]. Currently, the (bio)pharmaceutical industry is committed to improve the quality of their products while endeavouring a reduction of its production costs, which is intimately related to the necessity of improving their downstream processes, since this field is mainly dominated by chromatographic techniques [2] and are responsible for their current high-cost. Therefore, envisaging to develop new, cost-effective, efficient and sustainable techniques for proteins purification, polymer-polymer aqueous two-phase systems (ATPS) with ionic liquids (ILs) as adjuvants are herein proposed for the extraction and purification of IFNα2b from Escherichia coli BL21 cultures. The recombinant protein was produced in the form of inclusion bodies, followed by a dialysis step in order to solubilize the target biomolecule in a specific buffer, with the goal of maintaining the biomolecule stability. Afterwards, several polymers where studied as phase-forming components of ATPS, namely polyethylene glycol (PEG), polypropylene glycol (PPG), as well as Dextran. Furthermore, several ILs belonging to the cholinium, imidazolium, tetralkylammonium and tetralkylphosphonium families, were tested as co-adjuvants for ATPS formation in order to extract and purify of IFNα2b.publishe

New 1,3-diarylureas linked by C-C Suzuki coupling to the methyl 3-aminothieno[3,2-b]pyridine-2-carboxylate moiety: synthesis and fluorescence studies in solution and in lipid membranes

New six fluorescent 1,3-diarylureas linked by C-C Suzuki coupling to the 6-position of the methyl 3-aminothieno[3,2-b]pyridine-2-carboxylate moiety were prepared by reaction of the amino groups on the ortho or meta positions relative to the C-C bond of the Suzuki coupling products, with different para-substituted arylisocyanates (H, OMe, CN), in high to excellent yields. The fluorescence properties of the 1,3-diarylureas in solution and in lipid membranes of egg-yolk phosphatidylcholine (Egg-PC), dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylglycerol (DPPG) or dioctadecyldimethylammonium bromide (DODAB), with or without cholesterol (Ch), were studied. The six 1,3-diarylureas have reasonable fluorescence quantum yields in several solvents (between 0.02 and 0.69) and present a moderately solvent sensitive emission, but are not fluorescent in alcohols and water. The compounds bearing the arylurea moiety in the meta position relative to the C-C bond, especially with the OMe and CN substituents, present the better solvatochromic properties. Incorporation of the six compounds in lipid membranes indicates that all the compounds are deeply located in the hydrophobic region of the lipid bilayers, feeling the transition between the rigid gel phase and fluid phases.To the Foundation for the Science and Technology (FCT, Portugal) for inancial support to the NMR portuguese network (PTNMR, Bruker Avance III 400-Univ. Minho). To the FCT and FEDER (European Fund for Regional Development)-COMPETE-QREN-EU for financial support to the Research Centres, CQ/UM [PEst-C/QUI/UI0686/2011 (FCOMP-01-0124-FEDER-022716)] and CFUM [PEst-C/FIS/UI0607/2011 (F-COMP-01-0124-FEDER-022711)], and to the research projects PTDC/QUI/81238/2006 (FCOMP-01-0124-FEDER-007467) (photophysical studies) and PTDC/QUI-QUI/111060/2009 (F-COMP-01-0124-FEDER-015603) (organic synthesis)

Digestive enzyme activity and nutrient digestibility in meagre (Argyrosomus regius) fed increasing levels of black soldier fly meal (Hermetia illucens)

The effect of partially replacing fishmeal (FM) by black soldier fly larvae meal (Hermetia meal--HM) in meagre (Argyrosomus regius) diet was evaluated for nutrient digestibility and digestive enzyme activity. For that purpose, triplicate groups of fish (18.0 ± 0.02 g) were fed during 48 days either a control diet (CTR), without HM, or one of three diets including 100, 200 and 300 g/kg of HM, replacing 17, 35 and 52% of FM, respectively. Apparent digestibility coefficients (ADCs) of dry matter, energy, protein, lysine, isoleucine, leucine, phenylalanine, alanine, glutamate, glycine and serine presented a decreasing response with increased HM inclusion. Chitin ADC was null, independent of dietary HM inclusion. Total alkaline protease activity increased while trypsin activity decreased with dietary HM inclusion. No intestinal chitinolytic activity was detected. Intestinal alkaline protease zymogram revealed nine bands with proteolytic activity against casein, with molecular weights ranging between 15 and 75 kDa. Anti-protease activity in the intestine was not affected by dietary inclusion of HM compared to the CTR diet. Overall, it is concluded that replacement of up to 17% FM with HM (100 g/kg HM inclusion level) in meagre diets has no major adverse effects in diet digestibility and digestive enzyme activity