Structured emulsions as butter substitutes: effects on physicochemical and sensory attributes of shortbread cookies.

BACKGROUND: Reformulation of foods products to reduce total and saturated fats while maintaining acceptable structure, texture and mouthfeel poses an important challenge to the food industry. In this work, the use of structured emulsions (fibre-induced oil-in-water biphasic systems with reduced total and saturated fats) is proposed to replace butter in shortbread cookies. RESULTS: Use of structured emulsions resulted in softer dough that was still workable using a traditional process. Shortbread cookies containing structured emulsions were harder and paler than the butter control but had a significantly reduced saturated fat content. They also received promising scores in the sensory analysis in terms of texture and overall acceptability, despite the butter product still being the preferred sample. CONCLUSION: The results of this study indicated that structured emulsions represent a good solution to produce nutritionally improved shortbreads. Optimization of the structured emulsion formulation can provide further improvement of the nutritional, sensory and physicochemical properties of shortbread cookies. © 2018 Society of Chemical Industry.

Ubiquitin mediates the physical and functional interaction between human DNA polymerases η and ι.

Human DNA polymerases η and ι are best characterized for their ability to facilitate translesion DNA synthesis (TLS). Both polymerases (pols) co-localize in 'replication factories' in vivo after cells are exposed to ultraviolet light and this co-localization is mediated through a physical interaction between the two TLS pols. We have mapped the polη-ι interacting region to their respective ubiquitin-binding domains (UBZ in polη and UBM1 and UBM2 in polι), and demonstrate that ubiquitination of either TLS polymerase is a prerequisite for their physical and functional interaction. Importantly, while monoubiquitination of polη precludes its ability to interact with proliferating cell nuclear antigen (PCNA), it enhances its interaction with polι. Furthermore, a polι-ubiquitin chimera interacts avidly with both polη and PCNA. Thus, the ubiquitination status of polη, or polι plays a key regulatory function in controlling the protein partners with which each polymerase interacts, and in doing so, determines the efficiency of targeting the respective polymerase to stalled replication forks where they facilitate TLS.

Physicochemical, sensory properties and starch in vitro digestion of gluten-free breads.

The physicochemical (volume, crumb grain and color), sensory and starch in vitro digestion properties were investigated in four gluten-free breads, produced using gluten-free commercial mixes. Different mixes' formulations and processing parameters led to the production of breads with different crumb grain, specific volume, crust and crumb color, which affected product's sensory acceptability. Generally, more heterogeneous and coarser crumb grain and darker color were more appreciated by the judges. Nutritional composition of gluten-free breads differed mainly for starch and lipids contents, due to a wide variety of ingredients used in breads formulations. Starch availability to hydrolytic enzymes in the gluten-free breads was not influenced by variation in ingredients and nutritional composition, but it was related to the physical structure in terms of specific volume. The higher was the specific volume, the higher was the starch-digested fraction.

Residues Arg283, Arg285, and Ile287 in the nucleotide binding pocket of bovine viral diarrhea virus NS5B RNA polymerase affect catalysis and fidelity.

Residues Arg283, Arg285, and Ile287 are highly conserved amino acids in bovine viral diarrhea virus RNA polymerase (BVDV RdRp) and RdRps from related positive-strand RNA viruses. This motif is an important part of the binding pocket for the nascent RNA base pair during initiation and elongation. We found that replacement of the arginines with alanines or more conserved lysines or replacement of isoleucine with alanine or valine alters the ability of the mutant RdRps to incorporate ribonucleotides efficiently. The reduced RdRp activity stems from both decreased ribonucleotide binding and decreased catalytic efficiency in both primer-dependent and de novo initiation, as shown by kinetic studies. In line with other studies on flaviviral RdRps, our data suggest that Arg283 and Ile287 may be implicated in ribonucleotide binding and positioning of the template base in the active site. Arg285 appears to be involved directly in the selection of cognate nucleotide. The findings for Arg285 and Ile287 mutants also agree with similar data from picornavirus RdRps.

Defensive functioning in MtF and FtM transsexuals.

In spite of the potential clinical utility of defense mechanisms in the assessment of gender identity disorder patients as candidates to sex reassignment surgery, there is paucity of research in this field. The aim of the present study is therefore to ascertain whether the defensive profile of MtF and FtM transsexuals seeking sex reassignment surgery can be defined more primitive, immature and maladaptive than that of the two control groups. We compared the defensive profiles as assessed through the REM-71 (Steiner et al., 2001) of 104 MtF transsexuals, 46 FtM transsexuals and two control groups of males and females. Our results show that MtF transsexuals present an overall more primitive defensive array than that of both control groups, while FtMs show a profile not dissimilar from that of both control groups. Our results support the hypothesis that MtF transsexuals are characterized by higher proneness to psychopathology than the general population and show a more immature level of psychological functioning than FtM transsexuals.

DNA polymerase switching: effects on spontaneous mutagenesis in Escherichia coli.

Escherichia coli possesses five known DNA polymerases (pols). Pol III holoenzyme is the cell's main replicase, while pol I is responsible for the maturation of Okazaki fragments and filling gaps generated during nucleotide excision repair. Pols II, IV and V are significantly upregulated as part of the cell's global SOS response to DNA damage and under these conditions, may alter the fidelity of DNA replication by potentially interfering with the ability of pols I and III to complete their cellular functions. To test this hypothesis, we determined the spectrum of rpoB mutations arising in an isogenic set of mutL strains differentially expressing the chromosomally encoded pols. Interestingly, mutagenic hot spots in rpoB were identified that are susceptible to the actions of pols I-V. For example, in a recA730 lexA(Def) mutL background most transversions were dependent upon pols IV and V. In contrast, transitions were largely dependent upon pol I and to a lesser extent, pol III. Furthermore, the extent of pol I-dependent mutagenesis at one particular site was modulated by pols II and IV. Our observations suggest that there is considerable interplay among all five E. coli polymerases that either reduces or enhances the mutagenic load on the E. coli chromosome.

Effect of added ingredients on water status and physico-chemical properties of tomato sauce.

Different ingredients (guar, xanthan, carboxy methyl cellulose, locust bean gums, potato fiber, milk, potato and soy proteins) were added to tomato sauce to investigate their effect on its physico-chemical properties. The products were characterized in terms of colour, rheological properties (Bostwick consistency, flow behavior and consistency coefficient), water status (water activity, moisture content) and molecular mobility by 1H Nuclear Magnetic Resonance (NMR). Water activity was significantly decreased only by the addition of potato fiber. Xanthan, locust bean, guar and carboxy methyl cellulose significantly enhanced Bostwick consistency and consistency coefficient. Type of ingredient and concentration significantly affected 1H NMR mobility indicators. Principal component analysis (PCA) indicated that only 1H NMR mobility parameters were able to differentiate the effect of milk protein, xanthan and potato fiber on tomato sauce properties. The information collected in this work provides information to intelligently modulate tomato sauce attributes and tailor its properties for specific applications.

The use of potato fibre to improve bread physico-chemical properties during storage.

Bread staling reduction is a very important issue for the food industry. A fibre with high water holding capacity, extracted from potato peel, was studied for its ability to reduce bread staling even if employed at low level (0.4 g fibre/100 g flour). Physico-chemical properties (water activity, moisture content, frozen water content, amylopectin retrogradation) and (1)H Nuclear Magnetic Resonance molecular mobility were characterised in potato fibre added bread over 7 days of storage. Potato fibre addition in bread slightly affected water activity and moisture content, while increased frozen water content and resulted in a softer bread crumb, more importantly when the optimal amount of water was used in the formulation. Potato fibre also reduced (1)H NMR molecular mobility changes in bread crumb during storage. Potato fibre addition in bread contributed to reduce bread staling.

Effect of water and gluten on physico-chemical properties and stability of ready to eat shelf-stable pasta.

A multi-analytical and multi-dimensional approach was used to investigate the effect of moisture and gluten on physico-chemical properties of shelf-stable ready to eat (RTE) pasta. Moisture and frozen water contents were not affected by formulation nor storage time. Hardness and retrograded amylopectin significantly increased during storage in all samples, more markedly in pasta with the lowest moisture content. Higher amounts of water and gluten reduced pasta hardening and contributed to control RTE pasta quality. (1)H FID became steeper in all samples during storage, but no effect of high moisture and gluten levels was observed on the mobility of these protons. Three proton T2 populations were observed (population C, population D and population E). Population C and D were not resolved during all storage. (1)H T2 relaxation time of the most abundant population (population E) shifted to shorter times and the amount of protons increased during storage, more importantly in the samples with lower moisture and gluten content.

Expression, purification, and characterization of the Necator americanus aspartic protease-1 (Na-APR-1 (M74)) antigen, a component of the bivalent human hookworm vaccine.

Over 400 million people living in the world's poorest developing nations are infected with hookworms, mostly of the genus Necator americanus. A bivalent human hookworm vaccine composed of the Necator americanus Glutathione S-Transferase-1 (Na-GST-1) and the Necator americanus Aspartic Protease-1 (Na-APR-1 (M74)) is currently under development by the Sabin Vaccine Institute Product Development Partnership (Sabin PDP). Both monovalent vaccines are currently in Phase 1 trials. Both Na-GST-1 and Na-APR-1 antigens are expressed as recombinant proteins. While Na-GST-1 was found to express with high yields in Pichia pastoris, the level of expression of Na-APR-1 in this host was too low to be suitable for a manufacturing process. When the tobacco plant Nicotiana benthamiana was evaluated as an expression system, acceptable levels of solubility, yield, and stability were attained. Observed expression levels of Na-APR-1 (M74) using this system are ∼300 mg/kg. Here we describe the achievements and obstacles encountered during process development as well as characterization and stability of the purified Na-APR-1 (M74) protein and formulated vaccine. The expression, purification and analysis of purified Na-APR-1 (M74) protein obtained from representative 5 kg reproducibility runs performed to qualify the Na-APR-1 (M74) production process is also presented. This process has been successfully transferred to a pilot plant and a 50 kg scale manufacturing campaign under current Good Manufacturing Practice (cGMP) has been performed. The 50 kg run has provided a sufficient amount of protein to support the ongoing hookworm vaccine development program of the Sabin PDP.

Optimization and revision of the production process of the Necator americanus glutathione S-transferase 1 (Na-GST-1), the lead hookworm vaccine recombinant protein candidate.

Infection by the human hookworm Necator americanus is a leading cause of anemia and disability in the developing countries of Africa, Asia, and the Americas. In order to prevent childhood hookworm disease in resource poor settings, a recombinant vaccine is under development by the Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development, a Product Development Partnership (PDP). Previously, we reported on the expression and purification of a highly promising hookworm vaccine candidate, Na-GST-1, an N. americanus glutathione s-transferase expressed in Pichia pastoris (yeast), which led to production of 1.5 g of 95% pure recombinant protein at a 20L scale. (1) (,) (2) (,) (3) This yield and purity of Na-GST-1 was sufficient for early pilot manufacturing and initial phase 1 clinical testing. However, based on the number of doses which would be required to allow mass vaccination and a potential goal to deliver a vaccine as inexpensively as possible, a higher yield of expression of the recombinant antigen at the lowest possible cost is highly desirable. Here we report on modifications to the fermentation (upstream process) of the antigen expressed in P. pastoris, and to the purification (downstream process) of the recombinant protein that allowed for a 2-3-fold improvement in the final yield of Na-GST-1 purified protein. The major improvements included upstream process changes such as the addition of a sorbitol pulse and co-feed during methanol induction as well as an extension of the induction stage to approximately 96 hours; downstream process changes included modifying the UFDF to flat sheet with a 10 kDa Molecular Weight cut-off (MWCO), adjusting the capacity of an ion-exchange chromatography step utilizing a gradient elution as opposed to the original step elution, and altering the hydrophobic interaction chromatography conditions. The full process, as well as the purity and stability profiles of the target Na-GST-1, and its formulation on Alhydrogel(®), is described.

Biophysical and formulation studies of the Schistosoma mansoni TSP-2 extracellular domain recombinant protein, a lead vaccine candidate antigen for intestinal schistosomiasis.

A candidate vaccine to prevent human schistosomiasis is under development. The vaccine is comprised of a recombinant 9 kDa antigen protein corresponding to the large extracellular domain of a tetraspanin surface antigen protein of Schistosoma mansoni, Sm-TSP-2. Here, we describe the biophysical profile of the purified, recombinant Sm-TSP-2 produced in the yeast PichiaPink, which in preclinical studies in mice was shown to be an effective vaccine against intestinal schistosomiasis. Biophysical techniques including circular dichroism, intrinsic and extrinsic fluorescence and light scattering were employed to generate an empirical phase diagram, a color based map of the physical stability of the vaccine antigen over a wide range of temperatures and pH. From these studies a pH range of 6.0-8.0 was determined to be optimal for maintaining the stability and conformation of the protein at temperatures up to 25 °C. Sorbitol, sucrose and trehalose were selected as excipients that prevented physical degradation during storage. The studies described here provide guidance for maximizing the stability of soluble recombinant Sm-TSP-2 in preparation of its further development as a vaccine.

Expression at a 20L scale and purification of the extracellular domain of the Schistosoma mansoni TSP-2 recombinant protein: a vaccine candidate for human intestinal schistosomiasis.

A novel recombinant protein vaccine for human schistosomiasis caused by Schistosoma mansoni is under development. The Sm-TSP-2 schistosomiasis vaccine is comprised of a 9 kDa recombinant protein corresponding to the extracellular domain of a unique S. mansoni tetraspanin. Here, we describe the cloning and the expression of the external loop of Sm-TSP-2 recombinant protein secreted by Pichia Pink the process development at 20L scale fermentation, and the two-steps purification, which resulted in a protein recovery yield of 31% and a protein purity of 97%. The developed processes are suitable for the production of purified protein for subsequent formulation and Phase 1 clinical studies.

Evaluation of porous starch as a flavour carrier.

A commercial porous starch was evaluated for the use as a carrier for liquid flavours. Encapsulation trials performed with diacetyl showed a high initial load and good retention over time when more polar solvents commonly used in flavour creation were used. The physical interactions between the porous starch and solvents used in flavour creation were also studied. The glass transition temperature of the starch decreased upon addition of the polar solvents, ethanol and propylene glycol. Propylene glycol also produced an exothermic peak when mixed with porous starch, possibly due to the formation of complexes between the two components. Low resolution (1)H-NMR results suggested that a stronger interaction was established between more polar solvents and the porous starch, as indicated by a more marked decrease in relaxation times and proton diffusion coefficient of the solvents on adding porous starch.

Roadmap to developing a recombinant coronavirus S protein receptor-binding domain vaccine for severe acute respiratory syndrome.

A subunit vaccine, RBD-S, is under development to prevent severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV), which is classified by the US NIH as a category C pathogen. This vaccine is comprised of a recombinant receptor-binding domain (RBD) of the SARS-CoV spike (S) protein and formulated on alum, together with a synthetic glucopyranosyl lipid A. The vaccine would induce neutralizing antibodies without causing Th2-type immunopathology. Vaccine development is being led by the nonprofit product development partnership; Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development in collaboration with two academic partners (the New York Blood Center and University of Texas Medical Branch); an industrial partner (Immune Design Corporation); and Walter Reed Army Institute of Research. A roadmap for the product development of the RBD-S SARS vaccine is outlined with a goal to manufacture the vaccine for clinical testing within the next 5 years.

Characterization of the helicase activity and substrate specificity of Mycobacterium tuberculosis UvrD.

UvrD is a helicase that is widely conserved in gram-negative bacteria. A uvrD homologue was identified in Mycobacterium tuberculosis on the basis of the homology of its encoded protein with Escherichia coli UvrD, with which it shares 39% amino acid identity, distributed throughout the protein. The gene was cloned, and a histidine-tagged form of the protein was expressed and purified to homogeneity. The purified protein had in vitro ATPase activity that was dependent upon the presence of DNA. Oligonucleotides as short as four nucleotides were sufficient to promote the ATPase activity. The DNA helicase activity of the enzyme was only fueled by ATP and dATP. UvrD preferentially unwound 3'-single-stranded tailed duplex substrates over 5'-single-stranded ones, indicating that the protein had a duplex-unwinding activity with 3'-to-5' polarity. A 3' single-stranded DNA tail of 18 nucleotides was required for effective unwinding. By using a series of synthetic oligonucleotide substrates, we demonstrated that M. tuberculosis UvrD has an unwinding preference towards nicked DNA duplexes and stalled replication forks, representing the likely sites of action in vivo. The potential role of M. tuberculosis UvrD in maintenance of bacterial genomic integrity makes it a promising target for drug design against M. tuberculosis.