Gluten-Free Breads: The Gap Between Research and Commercial Reality.

The market for gluten-free products is steadily growing and gluten-free bread (GFB) keeps on being one of the most challenging products to develop. Although numerous research studies have worked on improving the manufacture of GFBs, some have adopted approaches far from commercial reality. This review analyzes the ingredient list and nutrition facts of 228 commercially available GFBs produced by different brands around the world. The results from studying the ingredient list of breads revealed that commercial breads do not tend to use a single starchy source or gluten replacer, but a combination of several ingredients to optimize bread quality. Maize, tuber starches, and rice flour were the main starchy sources. Regarding hydrocolloids, the most often included ingredients were hydroxypropyl methylcellulose, xanthan and guar gum, and psyllium. Proteins and sugars were added, respectively, in 81% and 87% of the commercial breads analyzed. Furthermore, it was found that vegetable oils were preferred over fats. A long list of ingredients was observed in commercial GFBs, with the presence of a wide range of additives, including acidifiers, emulsifiers, leavening agents, preservatives, and aromas or flavorings. Meanwhile, nutrition facts showed a lower protein and higher fat content for GFBs compared to a gluten-containing counterpart, with small differences for salt and sugar. This research expands the current knowledge on GFB manufacturing, giving a panoramic outlook on the current situation in the GFB market, and helping both scientists and gluten-free companies unify/identify common trends.

Ripe Banana Flour as a Source of Antioxidants in Layer and Sponge Cakes.

About one-fifth of all bananas harvested become culls that are normally disposed of improperly. However, ripe banana pulp contains significant amounts of fibre and polyphenol compounds as well as a high content of simple sugars (61.06 g/100 g), making it suitable for sucrose replacement in bakery products. This work studied the feasibility of incorporating ripe banana flour (20 and 40% of replacement) in cake formulation. Physical, nutritional and sensory attributes of sponge and layer cakes were evaluated. The inclusion of ripe banana flour generally led to an increased batter consistency that hindered cake expansion, resulting in a slightly lower specific volume and higher hardness. This effect was minimised in layer cakes where differences in volume were only evident with the higher level of replacement. The lower volume and higher hardness contributed to the decline of the acceptability observed in the sensory test. Unlike physical attributes, the banana flour inclusion significantly improved the nutritional properties of the cakes, bringing about an enhancement in dietary fibre, polyphenols and antioxidant capacity (up to a three-fold improvement in antioxidant capacity performance). Therefore, results showed that sugar replacement by ripe banana flour enhanced the nutritional properties of cakes, but attention should be paid to its inclusion level.

Degree of roasting of carob flour affecting the properties of gluten-free cakes and cookies.

Carob flour is a product rich in fibre obtained from by-products of the locust bean gum extraction processing. The flour is commercialised with different degrees of roasting in order to improve its organoleptic characteristics. In this study, carob flour with three different roasting degrees was used to replace rice flour (15%) in gluten-free cakes and cookies. The influence of this replacement was studied on the psychochemical characteristics and acceptability of the final products. The incorporation of carob flour increased the viscosity of cake batters and increased the solid elastic-like behaviour of the cookie doughs, indicating a stronger interaction among the formula ingredients. The inclusion of carob flour, with a low time of roasting, did not lead to any significant differences in the specific volume and hardness of the cakes, but reduced cake staling and the thickness and width of the cookies. Darker colours were obtained when carob flour was incorporated into the product. The acceptability of cakes was only reduced with the addition of highly roasted carob flour, while in the case of cookies there was a decline in the acceptability of all carob flour cookies, which was mostly perceived with the highest roasting degree, something mainly attributed to the bitter taste of the products.

Effect of extruded wheat flour as a fat replacer on batter characteristics and cake quality.

The effects of three levels of fat replacement (1/3, 2/3, and 3/3) by extruded flour paste and the effects of the presence of emulsifier on layer cake batter characteristics and final cake quality were studied. Replacement of oil by extruded flour paste modified the batter density and microscopy, reducing the number of air bubbles and increasing their size, while emulsifier incorporation facilitated air entrapment in batter. Emulsifier addition also increased the elastic and viscous moduli of the batter, while oil reduction resulted in a less structured batter. Emulsifier incorporation leads to good quality cakes, minimizing the negative effect of oil reduction, maintaining the volume and reducing the hardness of cakes. Furthermore, consumer acceptability of the reduced fat cakes was improved by the addition of emulsifier. Thus, the results confirmed the positive effect of partial oil substitution (up to 2/3) by extruded flour paste on the quality of reduced fat cakes when emulsifier was incorporated.

Limited sampling strategies for tacrolimus exposure (AUC0-24) prediction after Prograf(®) and Advagraf(®) administration in children and adolescents with liver or kidney transplants.

To develop limited sampling strategies (LSSs) to predict total tacrolimus exposure (AUC0-24 ) after the administration of Advagraf(®) and Prograf(®) (Astellas Pharma S.A, Madrid, Spain) to pediatric patients with stable liver or kidney transplants. Forty-one pharmacokinetic profiles were obtained after Prograf(®) and Advagraf(®) administration. LSSs predicting AUC0-24 were developed by linear regression using three extraction time points. Selection of the most accurate LSS was made based on the r(2) , mean error, and mean absolute error. All selected LSSs had higher correlation with AUC0-24 than the correlation found between C0 and AUC0-24 . Best LSS for Prograf(®) in liver transplants was C0_1.5_4 (r(2)  = 0.939) and for kidney transplants C0_1_3 (r(2)  = 0.925). For Advagraf(®) , the best LSS in liver transplants was C0_1_2.5 (r(2)  = 0.938) and for kidney transplants was C0_0.5_4 (r(2)  = 0.931). Excluding transplant type variable, the best LSS for Prograf(®) is C0-1-3 (r(2)  = 0.920) and the best LSS for Advagraf(®) was C0_0.5_4 (r(2)  = 0.926). Considering transplant type irrespective of the formulation used, the best LSS for liver transplants was C0_2_3 (r(2)  = 0.913) and for kidney transplants was C0_0.5_4 (r(2)  = 0.898). Best LSS, considering all data together, was C0_1_4 (r(2)  = 0.898). We developed several LSSs to predict AUC0-24 for tacrolimus in children and adolescents with kidney or liver transplants after Prograf(®) and/or Advagraf(®) treatment.

Conversion from Prograf to Advagraf in stable paediatric renal transplant patients and 1-year follow-up.

BACKGROUND: The conversion from Prograf to Advagraf on a 1:1 (mg:mg) basis has been questioned in light of the publication of studies showing a decrease in tacrolimus blood concentrations after the administration of Advagraf. METHODS: The bioavailability of Prograf and Advagraf was evaluated in an open-label conversion study in 21 stable renal transplant paediatric patients. Serial blood samples for determining tacrolimus levels were collected during a 24-h period before (on Prograf) and after (on Advagraf) conversion. Tacrolimus pharmacokinetic parameters were calculated using a non-compartmental approach and the relative bioavailability calculated. Clinical and analytical data were obtained at 30, 90, 180 and 360 days after study enrolment. RESULTS: The mean ratio and 90 % confidence interval (CI) for peak plasma drug concentration (C(max)) and the area under the time-concentration curve during the first 24 h (AUC(0-24)) were 81.54 (95 % CI 71.6-92.87) and 87.19 (95 % CI 79.91-95.13), respectively. Renal glomerular filtration rate remained stable over the course of the follow-up. Two patients presented clinical events unrelated to tacrolimus. Tacrolimus levels decreased in the first month, the dose/level ratio increased between months 1 and 6 and slight dose adjustments were required during the follow-up period. CONCLUSIONS: Our results show that Advagraf bioequivalence cannot be ensured in this population. Significant changes in tacrolimus levels and dose were observed on long-term follow-up.

Optimizing Screw Speed and Barrel Temperature for Textural and Nutritional Improvement of Soy-Based High-Moisture Extrudates.

Soy remains the legume protein of excellence for plant-based meat alternatives due to its fiber-forming potential. In this study, protein-rich powders from soy protein isolate (SPI), concentrate (SPC), and their mixture (SPM) were thoroughly characterized for their proximate composition, nutritional quality, and physicochemical properties to understand their structuring behavior during high-moisture extrusion. SPI presented higher degrees of protein denaturation and aggregation, least gelation concentration and lower essential amino acid contents. Thus, an SPI:SPC combination (1:9 ratio, 70% protein) was extruded at three different screw speeds (300, 350, and 400 rpm) and two temperature profiles (120 and 140 °C maximum temperature). The effects of the processing parameters on the extrudates were evaluated for their appearance (fibrousness), texture (TPA, cutting force, and anisotropy), color, protein structure (FTIR), and trypsin inhibitors. Higher temperatures resulted in softer and darker extrudates, with increased visual and instrumental anisotropy. Increasing screw speeds led to softer and lighter extrudates, without a clear fibrousness effect. ß-sheet structures decreased and intermolecular aggregates (A1) increased after extrusion, especially at 140 °C, together with the formation of intramolecular aggregates (A2). Extrusion also significantly decreased the amount of trypsin inhibitors (>90%). This study demonstrates that extrusion parameters need to be carefully selected to achieve meat analogs with optimal textural and nutritional characteristics.

TaqMan-quantitative PCR assays applied in Neospora caninum knock-outs generated through CRISPR-Cas9 allow to determine the copy numbers of integrated dihydrofolate reductase-thymidylate synthase drug selectable markers.

As for many other organisms, CRISPR-Cas9 mediated genetic modification has gained increasing importance for the identification of vaccine candidates and drug targets in Neospora caninum, an apicomplexan parasite causing abortion in cattle and neuromuscular disease in dogs. A widely used approach for generating knock-out (KO) strains devoid of virulence factors is the integration of a drug selectable marker such as mutated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) into the target gene, thus preventing the synthesis of respective protein and mediating resistance to pyrimethamine. However, CRISPR-Cas9 mutagenesis is not free of off-target effects, which can lead to integration of multiple mdhfr-ts copies into other sites of the genome. To determine the number of integrated mdhfr-ts in N. caninum, a duplex quantitative TaqMan PCR was developed. For this purpose, primers were designed that amplifies a 106 bp fragment from wild-type (WT) parasites corresponding to the single copy wtdhfrs-ts gene, as well as the mutated mdhfrs-ts present in KO parasites that confers resistance and were used simultaneously with primers amplifying the diagnostic NC5 gene. Thus, the dhfr-ts to NC5 ratio should be approximately 1 in WT parasites, while in KO parasites with a single integrated mdhrf-ts gene this ratio is doubled, and in case of multiple integration events even higher. This approach was applied to the Neospora KO strains NcΔGRA7 and NcΔROP40. For NcΔGRA7, the number of tachyzoites determined by dhfr-ts quantification was twice the number of tachyzoites determined by NC5 quantification, thus indicating that only one mdhfr-ts copy was integrated. The results obtained with the NcΔROP40 strain, however, showed that the number of dhfr-ts copies per genome was substantially higher, indicating that at least three copies of the selectable mdhfr-ts marker were integrated into the genomic DNA during gene editing by CRISPR-Cas9. This duplex TaqMan-qPCR provides a reliable and easy-to-use tool for assessing CRISPR-Cas9 mediated mutagenesis in WT N. caninum strains.

Periodic gastroenterology and hepatology meetings with primary care. Reasons for consultation.

INTRODUCTION: care overload, aging of population, and increased chronic diseases lead to increased referrals from primary care, which may sometimes overload the health system. Thus, different interventions have been carried out attempting to improve these aspects. OBJECTIVES: to assess the most frequent causes of consultation of general physicians, both in joint consultations and clinical sessions held jointly with specialist consultant in primary care, in the urban and rural setting, and the influence on referrals to first consultations of gastroenterology. MATERIAL AND METHODS: a mainly training type of intervention was carried out, consisting of regular meetings in both urban and rural primary care center, to perform joint consultations and clinical sessions on patients and topics related to the specialty of gastroenterology. The intervention period (divided in two subperiods) was compared with a control period. RESULTS: most reasons for consultation were those corresponding to lower gastrointestinal tract, followed by liver disease and upper gastrointestinal tract. Significant differences were only found in distribution of diagnoses between the two centers in joint consultations. There was a relative (percent) decrease in referrals at the global level in both subperiods, only significant in the first (51.45 %), as well as in rural setting (45.24 %). CONCLUSION: common consultations motifs were similar in urban and rural settings, with some relevance of lower gastrointestinal tract disease. Most of them can be solved at primary care, with the help of consultant specialist. There is impact on referrals to the outpatient first consultations of gastroenterology, mainly in rural setting.

Compositional Attributes of Blue Lupin (Lupinus angustifolius) Seeds for Selection of High-Protein Cultivars.

Lupin is a promising protein source with a high protein concentration. Breeding efforts have resulted in the development of varieties low in quinolizidine alkaloids. The objective of this work was to evaluate 22 different blue lupin genotypes for a high protein concentration and low content of antinutritional alkaloids. These genotypes were grown under uniform controlled environmental and soil conditions, and the harvested seeds were evaluated for their composition. The low phosphorus content confirmed that the phytic acid presence was low in lupin, especially compared to other legumes. Furthermore, some of the varieties had less than 200 ppm alkaloids. Lupin proteins were rich in leucine and lysine, with the lowest amino acid concentration being methionine. There were significant differences in the protein concentration and recovery. This work demonstrated that an approach for selection of genotypes should be based on not only agronomic yields but also nutritional phenotypes, driving better decision making on future varietal selection.

Preparation and characterization of self-standing biofilms from compatible pectin/starch blends: Effect of pectin structure.

Pectin structure-miscibility-functionality relationships in starch films remain unknown. In this study, five citrus pectins (CPs) with 17 to 63 % of degree of methyl esterification (DM) and sugar beet pectin (SBP, rich in acetyl moieties and rhamnogalacturonan-I domains) were investigated for composition and structure and, further, blended with pea starch (3:1 starch-pectin weight ratio) to fabricate self-standing films. The incorporation of pectin resulted in a two- to three-fold increase in tensile strength and Young's modulus (up to 52.2 and 1837 MPa, respectively, using CP with low DM) without compromising elongation at break. Starch-SBP films presented the lowest strength among pectin films. Lower film moisture and water vapor permeability were attained with CP of high DM, or with SBP, whereas surface wettability was explained by counteracting factors affecting film compositional heterogeneity. Films made with high methoxyl CP, or with SBP, showed lower overall H-bonding (FTIR) and starch crystallinity (XRD). A DM above 57 % negatively affected the mixing and interfacial adhesion of pectin with starch, as shown by Attenuated Total Reflection-FTIR imaging. Pectins with the lowest purity, presumably with the greatest content in xyloglucan, as suggested by HPAEC, presented ~20 % higher elongation at break than the other films.

Immunization with a Multivalent Listeria monocytogenes Vaccine Leads to a Strong Reduction in Vertical Transmission and Cerebral Parasite Burden in Pregnant and Non-Pregnant Mice Infected with Neospora caninum.

Neospora caninum is an apicomplexan parasite that causes abortion and stillbirth in cattle. We employed the pregnant neosporosis mouse model to investigate the efficacy of a modified version of the attenuated Listeria monocytogenes vaccine vector Lm3Dx_NcSAG1, which expresses the major N. caninum surface antigen SAG1. Multivalent vaccines were generated by the insertion of gra7 and/or rop2 genes into Lm3Dx_NcSAG1, resulting in the double mutants, Lm3Dx_NcSAG1_NcGRA7 and Lm3Dx_NcSAG1_NcROP2, and the triple mutant, Lm3Dx_NcSAG1_NcGRA7_NcROP2. Six experimental groups of female BALB/c mice were inoculated intramuscularly three times at two-week intervals with 1 × 107 CFU of the respective vaccine strains. Seven days post-mating, mice were challenged by the subcutaneous injection of 1 × 105N. caninum NcSpain-7 tachyzoites. Non-pregnant mice, dams and their offspring were observed daily until day 25 post-partum. Immunization with Lm3Dx_NcSAG1 and Lm3Dx_NcSAG1_NcGRA7_NcROP2 resulted in 70% postnatal pup survival, whereas only 50% and 58% of pups survived in the double mutant-vaccinated groups. Almost all pups had died at the end of the experiment in the infection control. The triple mutant was the most promising vaccine candidate, providing the highest rate of protection against vertical transmission (65%) and CNS infection. Overall, integrating multiple antigens into Lm3Dx_SAG1 resulted in lower vertical transmission and enhanced protection against cerebral infection in dams and in non-pregnant mice.

Whole-transcriptome analysis reveals virulence-specific pathogen-host interactions at the placenta in bovine neosporosis.

Research on bovine neosporosis has achieved relevant milestones, but the mechanisms underlying the occurrence of foetal death or protection against foetal death remain unclear. In a recent study, placentas from heifers challenged with the high-virulence isolate Nc-Spain7 exhibited focal necrosis and inflammatory infiltrates as soon as 10 days post-infection (dpi), although parasite detection was minimal. These lesions were more frequent at 20 dpi, coinciding with higher rates of parasite detection and the occurrence of foetal death in some animals. In contrast, such lesions were not observed in placentas from animals infected with the low-virulence isolate Nc-Spain1H, where the parasite was detected only in placenta from one animal at 20 dpi. This work aimed to study which mechanisms are triggered in the placentas (caruncles and cotyledons) of these pregnant heifers at early stages of infection (10 and 20 dpi) through whole-transcriptome analysis. In caruncles, infection with the high-virulence isolate provoked a strong proinflammatory response at 10 dpi. This effect was not observed in heifers infected with the low-virulence isolate, where IL-6/JAK/STAT3 signalling and TNF-alpha signalling via NF-κB pathways were down-regulated. Interestingly, the expression of E2F target genes, related to restraining the inflammatory response, was higher in these animals. At 20 dpi, more pronounced proinflammatory gene signatures were detectable in heifers infected with the high-virulence isolate, being more intense in heifers carrying dead fetuses. However, the low-virulence isolate continued without activating the proinflammatory response. In cotyledons, the response to infection with the high-virulence isolate was similar to that observed in caruncles; however, the low-virulence isolate induced mild proinflammatory signals at 20 dpi. Finally, a deconvolutional analysis of gene signatures from both placentome tissues revealed a markedly higher fraction of activated natural killers, M1 macrophages and CD8+ T cells for the high-virulence isolate. Therefore, our transcriptomic analysis supports the hypothesis that an intense immune response probably triggered by parasite multiplication could be a key contributor to abortion. Further studies are required to determine the parasite effectors that govern the distinct interactions of high- and low-virulence isolates with the host, which could help elucidate the molecular processes underlying the pathogenesis of neosporosis in cattle.

Characterization of Neospora caninum virulence factors NcGRA7 and NcROP40 in bovine target cells.

Bovine neosporosis is one of the major causes of reproductive failure in cattle worldwide, and differences in virulence between isolates have been widely shown. However, the molecular basis and mechanisms underlying virulence in Neospora caninum are mostly unknown. Recently, we demonstrated the involvement of NcGRA7 and NcROP40 in the virulence of N. caninum in a pregnant murine model using single knockout mutants in these genes generated by CRISR/Cas9 technology. In this study, the role of these proteins was investigated in two in vitro models using bovine target cells: trophoblast (F3 cell line) and monocyte-derived macrophages (BoMØ). The proliferation capacity of the single knockout mutant parasites was compared to the wild-type strain, the Nc-Spain7 isolate, using both cell populations. For the bovine trophoblast, no differences were observed in the growth of the defective parasites compared to the wild-type strain, neither in the proliferation kinetics nor in the competition assay. However, in naïve BoMØ, a significant decrease in the proliferation capacity of the mutant parasites was observed from 48 h pi onwards. Stimulation of BoMØ with IFN-γ showed a similar inhibition of tachyzoite growth in defective and wild-type strains in a dose-dependent manner. Finally, BoMØ infected with knockout parasites showed higher expression levels of TLR3, which is involved in pathogen recognition. These results suggest that NcGRA7 and NcROP40 may be involved in the manipulation of innate immune defense mechanisms against neosporosis and confirm the usefulness of the BoMØ model for the evaluation of N. caninum virulence mechanisms. However, the specific functions of these proteins remain unknown, opening the way for future research.

Electroconvulsive therapy use for refractory status epilepticus in an implantable vagus nerve stimulation patient: A case report.

Introduction: Status epilepticus (SE) has a mortality rate of 20 to 50%, with acute symptomatic SE having a higher risk compared to chronic SE. Electroconvulsive therapy (ECT) has been utilized for the treatment of refractory SE with a success rate estimate of 57.9%. There are no known reported cases of concomitant use of vagus nerve stimulation (VNS) and ECT for the treatment of super refractory SE (SRSE) available in the literature. Case description: We present a 44-year-old female with a history of developmental delay, epilepsy, an implantable VNS for 6 years, and traumatic brain injury with subsequent hygroma who presented with progressive aphasia, declining mental status, and daily generalized seizures lasting up to 20 min. Seizures had increased from her baseline of one seizure per day controlled with topiramate 200 mg three times daily and lamotrigine 400 mg twice daily. She was diagnosed with SRSE after being intubated and placed on eight anti-epileptic drugs (AEDs) that failed to abort SE. ECT was attempted to terminate SE. Due to a prior right craniotomy with subsequent right hygroma, eight treatments of ECT were performed over three sessions using a right anterior, left temporal (RALT) and subsequently a bitemporal electrode placement. The VNS remained active throughout treatment. Various ECT dosing parameters were attempted, varying pulse width and frequency. Although ECT induced mild transient encephalographic (EEG) changes following ECT stimulations, it was unable to terminate SE. Discussion: This case describes various treatment strategies, constraints, and device limitations when using ECT for the treatment of SE. With wide variability in efficacy rates of ECT in the treatment of SE in the literature, successful and unsuccessful cases offer information on optimizing ECT total charge dose and parameters that yielded success. This case demonstrates an instance of ECT inefficacy in the treatment of SRSE. Here, we discuss the rationale behind the various ECT settings that were selected, and constraints arising from the antiepileptic burden, VNS, and intrinsic limitations of the ECT device itself.

Pregelatinized Drum-Dried Wheat Starch of Different Swelling Behavior as Clean Label Oil Replacer in Oil-in-Water Emulsions.

Drum-drying results in pregelatinized starch with relatively low starch fragmentation and a great ability to absorb water and swell at room temperature. However, the effect of the degree of cold particle swelling and the thickening potential of drum-dried starch on its suitability as oil replacer in low-fat oil-in-water emulsions has received little attention. In this work, the potential of three pregelatinized drum-dried starches with almost identical molecular structure (as measured by size exclusion chromatography) and Water Binding Capacity (WBC), but different swelling behavior, was investigated to replace up to 60% oil in a mayonnaise-like emulsion system. The microstructure, stability, and rheology of the oil-in-water emulsions were noticeably affected by the substitution of oil with a pregelatinized drum-dried starch paste. Specifically, reduced-fat emulsions presented smaller droplet-size, a higher consistency index and increased emulsion stability, especially against freeze-thaw cycles, compared to the control full-fat mayonnaise. Importantly, the differences in cold swelling behavior (rather than simply assessing WBC) greatly influenced the consistency index and stability of low-fat emulsions, and results showed that drum-dried starch particles with high swelling potential perform better as oil replacers.

Changes to fine structure, size and mechanical modulus of phytoglycogen nanoparticles subjected to high-shear extrusion.

This study aims to enhance the understanding of the structure of maize phytoglycogen nanoparticles, and the effect of shear scission on their architecture, radius, stiffness, and deformability. Compared to amylopectin, phytoglycogen had a lower A:B chain ratio, a lower number of chains per B chain, and a much higher number of Afingerprint chains. Phytoglycogen (Mw = 28.0 × 106 g/mol) was subjected to high-shear extrusion with varying Specific Mechanical Energies (SMEs) using different screw speeds, showing a maximum stable molecular weight Mw of ∼9.31 × 106 g/mol and a particle radius R reduction of 36 %, with a corresponding 20 % increase in the average mass density. Atomic force microscopy force spectroscopy revealed that nanoparticles extruded at the lowest SME (122 Wh/kg) exhibited a 20 % increase in Young's modulus. Higher SME values (up to 488 Wh/kg) resulted in an overall decrease in stiffness without further significant reductions in radius.

NcGRA7 and NcROP40 Play a Role in the Virulence of Neospora caninum in a Pregnant Mouse Model.

The intraspecific variability among Neospora caninum isolates in their in vitro behaviour and in vivo virulence has been widely studied. In particular, transcriptomic and proteomic analyses have shown a higher expression/abundance of specific genes/proteins in high-virulence isolates. Consequently, the dense granule protein NcGRA7 and the rhoptry protein NcROP40 were proposed as potential virulence factors. The objective of this study was to characterize the role of these proteins using CRISPR/Cas9 knockout (KO) parasites in a well-established pregnant BALB/c mouse model of N. caninum infection at midgestation. The deletion of NcGRA7 and NcROP40 was associated with a reduction of virulence, as infected dams displayed milder clinical signs, lower parasite burdens in the brain, and reduced mortality rates compared to those infected with the wild-type parasite (Nc-Spain7). Specifically, those infected with the NcGRA7 KO parasites displayed significantly milder clinical signs and a lower brain parasite burden. The median survival time of the pups from dams infected with the two KO parasites was significantly increased, but differences in neonatal mortality rates were not detected. Overall, the present study indicates that the disruption of NcGRA7 considerably impairs virulence in mice, while the impact of NcROP40 deletion was more modest. Further research is needed to understand the role of these virulence factors during N. caninum infection.

Metallothionein expression in chloroplasts enhances mercury accumulation and phytoremediation capability.

Genetic engineering to enhance mercury phytoremediation has been accomplished by expression of the merAB genes that protects the cell by converting Hg[II] into Hg[0] which volatilizes from the cell. A drawback of this approach is that toxic Hg is released back into the environment. A better phytoremediation strategy would be to accumulate mercury inside plants for subsequent retrieval. We report here the development of a transplastomic approach to express the mouse metallothionein gene (mt1) and accumulate mercury in high concentrations within plant cells. Real-time PCR analysis showed that up to 1284 copies of the mt1 gene were found per cell when compared with 1326 copies of the 16S rrn gene, thereby attaining homoplasmy. Past studies in chloroplast transformation used qualitative Southern blots to evaluate indirectly transgene copy number, whereas we used real-time PCR for the first time to establish homoplasmy and estimate transgene copy number and transcript levels. The mt1 transcript levels were very high with 183,000 copies per ng of RNA or 41% the abundance of the 16S rrn transcripts. The transplastomic lines were resistant up to 20 µm mercury and maintained high chlorophyll content and biomass. Although the transgenic plants accumulated high concentrations of mercury in all tissues, leaves accumulated up to 106 ng, indicating active phytoremediation and translocation of mercury. Such accumulation of mercury in plant tissues facilitates proper disposal or recycling. This study reports, for the first time, the use of metallothioneins in plants for mercury phytoremediation. Chloroplast genetic engineering approach is useful to express metal-scavenging proteins for phytoremediation.

The effect of extruded breadfruit flour on structural and physicochemical properties of beef emulsion modeling systems.

The objective was to determine structural and physicochemical properties of beef emulsion modeling systems prepared with native breadfruit flour and four different extruded breadfruit flours. Extrusion conditions for the flours were summarized as two different specific mechanical energies (74 or 145 kJ/kg) and four unique melt temperatures (83 °C, 100 °C, 105 °C, or 126 °C). Meat emulsions formulated at 3% replacement of beef with native or extruded breadfruit flours were compared with control (no additional flour) formulations. Replacement of beef with breadfruit flour (either native or extruded) did not significantly change cooking loss or instrumental redness values of cooked meat emulsions. Interestingly, replacement of beef with the fully gelatinized extruded breadfruit flours altered viscosity during heating as indicated by lower values for storage modulus (44.75% to 62.53% decrease compared with control) and lower values for loss modulus (25.90% to 52.54% decrease compared with control). This resulted in meat emulsions with a significant reduction in textural hardness (28.78% to 37.62% decrease compared with control).