Fluorescent labeling of DNA : strategies, pitfalls and necessity for fluorescence microscopy investigations of gene therapy

Gene therapy is a field of research in which a huge amount of effort has been spent over the last two decades. To succeed, a correct therapeutic gene needs to be delivered into the cytoplasm of a cell for mRNA and nucleus of a cell for plasmid DNA (pDNA). To protect the nucleic acids, viral vectors and non-viral carriers have been used. Due to the many barriers that are encountered in the delivery, the design and evaluation of especially the non-viral vectors has room for improvement. To this end, fluorescence microscopy has proven to be a useful tool. In this thesis, the main goal was to study the degradation of pDNA with advanced microscopy. Since fluorescence microscopy requires a fluorescent tag on the molecules of interest, an overview of the possible nucleic acid labeling strategies was given. Attention was given to the effect the methods have on the intracellular processing. This is studied in detail for a frequently used random covalent labeling method. When using Lipofectamine, the transfection efficiency drops for high labeling densities, probably due to an increased hydrophobicity which causes a higher affinity for lipid structures and steric hindrance of the labels for transcriptional proteins. Alternative pDNA labeling strategies were also explored. Fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT) were evaluated for their ability to follow pDNA degradation. SPT could measure the degradation of pDNA in cell lysate after lipofection. Finally, the gene delivery potential of 3 polycationic amphiphilic β-cyclodextrins (CDs) was tested for pDNA and mRNA. Due to a lower cellular uptake, no transfection could be induced in the presence of serum. It was seen that the interactions of CDs with cellular cholesterol are likely blocked by the serum proteins. In conclusion, we have shown that the correct use of microscopy methods, and especially SPT, is valuable in the study and evaluation of barriers for non-viral nucleic acid carriers

Fluorescent labeling of plasmid DNA and mRNA : gains and losses of current labeling strategies

Live-cell imaging has provided the life sciences with insights into the cell biology and dynamics. Fluorescent labeling of target molecules proves to be indispensable in this regard. In this Review, we focus on the current fluorescent labeling strategies for nucleic acids, and in particular mRNA (mRNA) and plasmid DNA (pDNA), which are of interest to a broad range of scientific fields. By giving a background of the available techniques and an evaluation of the pros and cons, we try to supply scientists with all the information needed to come to an informed choice of nucleic acid labeling strategy aimed at their particular needs

Fluorescence-based quantification of messenger RNA and plasmid DNA decay kinetics in extracellular biological fluids and cell extracts

Extracellular and intracellular degradation of nucleic acids remains an issue in non-viral gene therapy. Understanding biodegradation is critical for the rational design of gene therapeutics in order to maintain stability and functionality at the target site. However, there are only limited methods available that allow determining the stability of genetic materials in biological environments. In this context, the decay kinetics of fluorescently labeled plasmid DNA (pDNA) and messenger RNA (mRNA) in undiluted biological samples (i.e., human serum, human ascites, bovine vitreous) and cell extracts is studied using fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT). It is demonstrated that FCS is suitable to follow mRNA degradation, while SPT is better suited to investigate pDNA integrity. The half-life of mRNA and pDNA is approximate to 1-2 min and 1-4 h in biological samples, respectively. The resistance against biodegradation drastically improves by complexation with lipid-based carriers. Taken together, FCS and SPT are able to quantify the integrity of mRNA and pDNA, respectively, as a function of time, both in the extracellular biological fluids and cell extracts. This in turn allows to focus on the important but less understood issue of nucleic acids degradation in more detail and to rationally optimize gene delivery system as therapeutics

Stealth monoolein-based nanocarriers for delivery of siRNA to cancer cells

While the delivery of small interfering RNAs (siRNAs) is an attractive strategy to treat several clinical con- ditions, siRNA-nanocarriers stability after intravenous administration is still a major obstacle for the development of RNA-interference based therapies. But, although the need for stability is well recognized, the notion that strong stabilization can decrease nanocarriers efficiency is sometimes neglected. In this work we evaluated two stealth functionalization strategies to stabilize the previously validated dioctade- cyldimethylammonium bromide (DODAB):monoolein (MO) siRNA-lipoplexes. The nanocarriers were pre- and post-pegylated, forming vectors with different stabilities in biological fluids. The stealth nanocarriers behavior was tested under biological mimetic conditions, as the production of stable siRNA-lipoplexes is determinant to achieve efficient intravenous siRNA delivery to cancer cells. Upon incubation in human serum for 2 h, by fluorescence Single Particle Tracking microscopy, PEG-coated lipo- plexes were found to have better colloidal stability as they could maintain a relatively stable size. In addi- tion, using fluorescence fluctuation spectroscopy, post-pegylation also proved to avoid siRNA dissociation from the nanocarriers in human serum. Concomitantly it was found that PEG-coated lipoplexes improved cellular uptake and transfection efficiency in H1299 cells, and had the ability to silence BCR-ABL, affecting the survival of K562 cells. Based on an efficient cellular internalization, good silencing effect, good siRNA retention and good col- loidal stability in human serum, DODAB:MO (2:1) siRNA-lipoplexes coated with PEG-Cer are considered promising nanocarriers for further in vivo validation. Statement of Significance This work describes two stealth functionalization strategies for the stabilization of the previously validated dioctadecyldimethylammonium bromide (DODAB):monoolein (MO) siRNA-lipoplexes. These nanocarriers are capable of efficiently incorporating and delivering siRNA molecules to cells in order to silence genes whose expression is implicated in a pathological condition. The main objective was to functionalize these nanocarriers with a coating conferring protection to siRNA in blood without compromising its efficient delivery to cancer cells, validating the potential of DODAB:MO (2:1) siRNA-lipoplexes as therapeutic vec- tors. We show that the stealth strategy is determinant to achieve a stable and efficient nanocarrier, and that DODAB:MO mixtures have a very promising potential for systemic siRNA delivery to leukemic cells.FEDER through POFC-COMPETE and by national funds from FCT I.P. through the strategic funding UID/BIA/04050/2013 (CBMA) and PEst-C/FIS/UI0607/2013 (CFUM) and PTDC/QUI/69795/2006. We thank the support of the Frame Work Program 7 of the European Commission: BIOCAPS (316265, FP7/REGPOT) and Xunta de Galicia, Spain (Agrupamento INBIOMED, Grupo con potencial crecimiento) reference IF/00498/2012, scholarship SFRH/BD/68588/2010. NanoDelivery-I&D em Bionanotecnologia, Lda. for access to their equipment

The impact of salt and alkali on gluten polymerization and quality of fresh wheat noodles

We investigated the impact of table (NaCl) and alkaline (kansui) salts on changes to the gluten network during fresh wheat noodle production and cooking. Noodle production did not markedly change the gluten structure. In contrast, cooking increased gluten's average molecular weight by disulfide bond formation or reshuffling as evidenced by the decrease of protein extractability. Addition of NaCl (0.5 up to 3.0 weight % on flour basis) to the recipe reduced the extent of gluten polymerization during cooking. Kansui (0.2 up to 1.5 weight %) increased intermolecular disulfide bond formation. Furthermore, amino acid analysis revealed that kansui induced the formation of dehydroalanine-derived cross-links lanthionine and lysinoalanine. Optimal firmness was observed for noodles containing either 0.2–1.5% kansui or 2.0% NaCl. However, the addition of kansui reduced noodle nutritional quality, and high levels of table (2.0–3.0%) or alkaline (1.0–1.5%) salt increased cooking losses.publisher: Elsevier articletitle: The impact of salt and alkali on gluten polymerization and quality of fresh wheat noodles journaltitle: Journal of Cereal Science articlelink: http://dx.doi.org/10.1016/j.jcs.2014.09.003 content_type: article copyright: Copyright © 2014 Elsevier Ltd. All rights reserved.status: publishe

Amaranth (Amaranthus spp.), buckwheat (Fagopyrum spp.), and quinoa (Chenopodium spp.) proteins: a food science and technology perspective

The use of pseudocereals as raw materials for developing nutritious food products is skyrocketing. Pseudocereals are dicotyledonous crops - unlike true cereals, which are monocotyledonous - with starch-rich seeds. The three major pseudocereals in terms of world production are amaranth, buckwheat, and quinoa. Apart from starch, they contain high levels of protein, dietary fiber, minerals, vitamins, and other bioactive constituents. Their proteins have well-balanced amino acid compositions, are more sustainable than those from animal sources, and can safely be incorporated in the diet of celiac patients. While prolamins and glutelins are the most important protein fractions of true cereals, the proteins of pseudocereals are predominantly albumins and globulins. The traditional Osborne fractionation scheme is not always rigorously followed for pseudocereals, particularly in the methods used to extract globulins and glutelins. It is also of note here that, in order to establish in-depth structure-function relationships, future research should aim at (i) unlocking the genome sequence of pseudocereal proteins and (ii) unraveling the primary, secondary, tertiary, and quaternary structures of amaranth, buckwheat, and quinoa proteins. Basic foaming, emulsifying, and gelling properties of pseudocereal proteins are often similar to or even better than those of industrially relevant protein sources such as casein, which makes them potential substitutes for animal proteins in many food applications. It is, however, imperative to emphasize that, in most studies concerning the functionality of pseudocereal proteins, the protein isolates were obtained by alkaline extraction and subsequent isoelectric precipitation at acidic pH. Such harsh extraction conditions provoke protein denaturation and thus alter the native protein conformation and, as a consequence, the techno-functional properties. Increasing knowledge on structural changes in the native proteins from amaranth, buckwheat, and quinoa during processing steps relevant to food production and the effect thereof on their techno-functional properties is essential to expand the prospects for exploitation of pseudocereals.status: accepte

Effect of temperature, time and wheat gluten moisture content on wheat gluten network formation during thermomolding

A plastic-like material can be obtained by thermomolding wheat gluten protein which consists of glutenin and gliadin. We studied the effect of molding temperature (130-170 °C), molding time (5-25 min) and initial wheat gluten moisture content (5.6-18.0%) on the gluten network. Almost no glutenins were extractable after thermomolding irrespective of the molding conditions. At the lowest molding temperature, the extractable gliadin content decreased with increasing molding times and moisture contents. This effect was more pronounced for the α- and γ-gliadins than for the ω-gliadins. Protein extractabilities under reducing conditions revealed that, at this molding temperature, the cross-linking was predominantly based on disulfide bonds. At higher molding temperatures, also non-disulfide bonds contributed to the gluten network. Decreasing cystine contents and increasing free sulfhydryl and dehydroalanine (DHA) contents with increasing molding temperatures and times revealed the occurrence of beta-elimination reactions during thermomolding. Under the experimental conditions, the DHA derived cross-link lanthionine (LAN) was detected in all gluten samples thermomolded at 150 and 170 °C. LAN was also formed at 130 °C for gluten samples containing 18.0% moisture. Degradation was observed at 150 °C for samples thermomolded from gluten with 18.0% moisture content or thermomolded at 170 °C for all moisture contents.status: publishe

Proteins of Amaranth (Amaranthus spp.), Buckwheat (Fagopyrum spp.), and Quinoa (Chenopodium spp.): A Food Science and Technology Perspective

© 2016 Institute of Food Technologists® There is currently much interest in the use of pseudocereals for developing nutritious food products. Amaranth, buckwheat, and quinoa are the 3 major pseudocereals in terms of world production. They contain high levels of starch, proteins, dietary fiber, minerals, vitamins, and other bioactives. Their proteins have well-balanced amino acid compositions, are more sustainable than those from animal sources, and can be consumed by patients suffering from celiac disease. While pseudocereal proteins mainly consist of albumins and globulins, the predominant cereal proteins are prolamins and glutelins. We here discuss the structural properties, denaturation and aggregation behaviors, and solubility, as well as the foaming, emulsifying, and gelling properties of amaranth, buckwheat, and quinoa proteins. In addition, the technological impact of incorporating amaranth, buckwheat, and quinoa in bread, pasta, noodles, and cookies and strategies to affect the functionality of pseudocereal flour proteins are discussed. Literature concerning pseudocereal proteins is often inconsistent and contradictory, particularly in the methods used to obtain globulins and glutelins. Also, most studies on protein denaturation and techno-functional properties have focused on isolates obtained by alkaline extraction and subsequent isoelectric precipitation at acidic pH, even if the outcome of such studies is not necessarily relevant for understanding the role of the native proteins in food processing. Finally, even though establishing in-depth structure–function relationships seems challenging, it would undoubtedly be of major help in the design of tailor-made pseudocereal foods.status: publishe

Conditions Governing Food Protein Amyloid Fibril Formation. Part II: Milk and Legume Proteins

Both intrinsic and extrinsic factors impact amyloid formation of food proteins. We here review the impact of various conditions and food constituents on amyloid fibrillation of milk and legume proteins. Much less is known about casein and legume protein amyloid-like fibril (AF) formation than about that of whey proteins such as β-lactoglobulin, α-lactalbumin and bovine serum albumin. Proteins of both sources are often studied after heating under strong acidic (pH < 3) conditions. The latter induces changes in protein conformation and often peptide hydrolysis. At higher pH values, alcohols, chaotropic and/or reducing agents induce the conformational changes required to enhance fibrillation. Different types of food proteins can impact each other’s fibrillation. Also, the presence of other food constituents can enhance or reduce it. No general conclusions on the mechanisms or impact of different food constituents on food proteins can be made. Optimal conditions for AF formation, i.e. heating for several days at low pH, are rare in food processing. However, this does not exclude the possibility of AF formation in food products. For example, slow cooking of hydrolyzed proteins may enhance it. Future research should focus on the prevalence of AFs in complex food systems or model systems relevant for food processing.status: publishe