Unraveling the differential mechanisms of revascularization promoted by MSCs & ECFCs from adipose tissue or umbilical cord in a murine model of critical limb-threatening ischemia.

BACKGROUND: Critical limb-threatening ischemia (CLTI) constitutes the most severe manifestation of peripheral artery disease, usually induced by atherosclerosis. CLTI patients suffer from high risk of amputation of the lower extremities and elevated mortality rates, while they have low options for surgical revascularization due to associated comorbidities. Alternatively, cell-based therapeutic strategies represent an effective and safe approach to promote revascularization. However, the variability seen in several factors such as cell combinations or doses applied, have limited their success in clinical trials, being necessary to reach a consensus regarding the optimal "cellular-cocktail" prior further application into the clinic. To achieve so, it is essential to understand the mechanisms by which these cells exert their regenerative properties. Herein, we have evaluated, for the first time, the regenerative and vasculogenic potential of a combination of endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) isolated from adipose-tissue (AT), compared with ECFCs from umbilical cord blood (CB-ECFCs) and AT-MSCs, in a murine model of CLTI. METHODS: Balb-c nude mice (n:32) were distributed in four different groups (n:8/group): control shams, and ischemic mice (after femoral ligation) that received 50 µl of physiological serum alone or a cellular combination of AT-MSCs with either CB-ECFCs or AT-ECFCs. Follow-up of blood flow reperfusion and ischemic symptoms was carried out for 21 days, when mice were sacrificed to evaluate vascular density formation. Moreover, the long-term molecular changes in response to CLTI and both cell combinations were analyzed in a proteomic quantitative approach. RESULTS: AT-MSCs with either AT- or CB-ECFCs, promoted a significant recovery of blood flow in CLTI mice 21 days post-ischemia. Besides, they modulated the inflammatory and necrotic related processes, although the CB group presented the slowest ischemic progression along the assay. Moreover, many proteins involved in the repairing mechanisms promoted by cell treatments were identified. CONCLUSIONS: The combination of AT-MSCs with AT-ECFCs or with CB-ECFCs promoted similar revascularization in CLTI mice, by restoring blood flow levels, together with the modulation of the inflammatory and necrotic processes, and reduction of muscle damage. The protein changes identified are representative of the molecular mechanisms involved in ECFCs and MSCs-induced revascularization (immune response, vascular repair, muscle regeneration, etc.).

Atherosclerotic Pre-Conditioning Affects the Paracrine Role of Circulating Angiogenic Cells Ex-Vivo.

In atherosclerosis, circulating angiogenic cells (CAC), also known as early endothelial progenitor cells (eEPC), are thought to participate mainly in a paracrine fashion by promoting the recruitment of other cell populations such as late EPC, or endothelial colony-forming cells (ECFC), to the injured areas. There, ECFC replace the damaged endothelium, promoting neovascularization. However, despite their regenerative role, the number and function of EPC are severely affected under pathological conditions, being essential to further understand how these cells react to such environments in order to implement their use in regenerative cell therapies. Herein, we evaluated the effect of direct incubation ex vivo of healthy CAC with the secretome of atherosclerotic arteries. By using a quantitative proteomics approach, 194 altered proteins were identified in the secretome of pre-conditioned CAC, many of them related to inhibition of angiogenesis (e.g., endostatin, thrombospondin-1, fibulins) and cell migration. Functional assays corroborated that healthy CAC released factors enhanced ECFC angiogenesis, but, after atherosclerotic pre-conditioning, the secretome of pre-stimulated CAC negatively affected ECFC migration, as well as their ability to form tubules on a basement membrane matrix assay. Overall, we have shown here, for the first time, the effect of atherosclerotic factors over the paracrine role of CAC ex vivo. The increased release of angiogenic inhibitors by CAC in response to atherosclerotic factors induced an angiogenic switch, by blocking ECFC ability to form tubules in response to pre-conditioned CAC. Thus, we confirmed here that the angiogenic role of CAC is highly affected by the atherosclerotic environment.

Differences in dietary and lifestyle habits between pregnant women with small fetuses and appropriate-for-gestational-age fetuses.

AIM: The purpose of this study was to determine differences in lifestyle and dietary habits between pregnant women with small for gestational age (SGA) fetuses and those appropriate for gestational age (AGA). METHODS: This cross-sectional, prospective study was conducted over 14 months and included 46 mothers with SGA and 81 with AGA fetuses. Fetal growth was assessed by a routine third trimester scan. Participants completed questionnaires regarding dietary habits, lifestyle behavior and sociodemographic characteristics during the third trimester. RESULTS: Mothers maintaining a Mediterranean-type diet and lifestyle were more likely to have an AGA fetus (P < 0.05). The same number of women in both groups ate a vegetarian diet. Women in the SGA group had a lower intake of certain micronutrients: carotene, folic acid, iron, potassium and magnesium (P < 0.05). Women in the AGA group ate more vegetables, especially green beans, carrots, lettuce and oranges (P < 0.05). Bread, pasta, cakes and jam were also more frequently consumed by the AGA group (P < 0.05). Mothers in the SGA group drank more cola (P < 0.05), while mothers in the AGA group drank more diet cola and wine (P < 0.05). Women in the SGA group smoked more cigarettes per day (P < 0.05). CONCLUSIONS: Mothers of SGA fetuses had a different nutritional intake and cigarette smoking habits compared with mothers of AGA fetuses. No other differentiating lifestyle habits were observed between the groups. Thus, intrauterine growth conditions might be improved by the endorsement of certain dietary habits during pregnancy.

Placental 11B-Hydroxysteroid Dehydrogenase Type 2 mRNA Levels in Intrauterine Growth Restriction versus Small-for-Gestational-Age Fetuses.

INTRODUCTION: The objective of this study was to evaluate placental 11B-hydroxysteroid dehydrogenase type 2 (11B-HSD-2) mRNA levels in intrauterine growth-restricted fetuses (IUGR) as compared with small-for-gestational-age (SGA) fetuses according to clinical criteria. MATERIAL AND METHODS: Placental levels of 11B-HSD-2 mRNA levels were measured in SGA (birth weight <10th centile) and gestational-age-matched, appropriate-for-gestational-age (AGA) births. SGA was classified as IUGR (birth weight <3rd centile or <10th percentile with abnormal uterine artery Doppler or cerebroplacental ratio) or non-IUGR SGA. After RNA extraction, mRNA levels were determined by reverse transcription and quantitative PCR. RESULTS: A total of 38 placentas were analyzed (20 AGA and 18 SGA). Among the SGA pregnancies, 13 qualified as IUGR. The activity of 11B-HSD-2 in IUGR pregnancies [0.105 (SD 0.328)] was significantly reduced compared to non-IUGR SGA [0.304 (SD 0.261); p = 0.018] and AGA [0.294 (SD 0.328); p = 0.001]. These differences remained significant after adjusting for potential confounders (such as smoking or maternal cortisol levels). Activity levels did not significantly differ between non-IUGR SGA and AGA. DISCUSSION: IUGR fetuses had reduced 11B-HSD-2 activity in comparison with SGA and normally grown fetuses. This finding provides opportunities to develop new placental biomarkers for the phenotypic characterization of fetal smallness.

Distinct serum proteome profiles associated with collagen-induced arthritis and complete Freund's adjuvant-induced inflammation in CD38⁻/⁻ mice: The discriminative power of protein species or proteoforms.

Collagen-type-II-induced arthritis (CIA) is an autoimmune disease, which involves a complex host systemic response including inflammatory and autoimmune reactions. CIA is milder in CD38(-/-) than in wild-type (WT) mice. ProteoMiner-equalized serum samples were subjected to 2D-DiGE and MS-MALDI-TOF/TOF analyses to identify proteins that changed in their relative abundances in CD38(-/-) versus WT mice either with arthritis (CIA(+) ), with no arthritis (CIA(-) ), or with inflammation (complete Freund's adjuvant (CFA)-treated mice). Multivariate analyses revealed that a multiprotein signature (n = 28) was able to discriminate CIA(+) from CIA(-) mice, and WT from CD38(-/-) mice within each condition. Likewise, a distinct multiprotein signature (n = 16) was identified which differentiated CIA(+) CD38(-/-) mice from CIA(+) WT mice, and lastly, a third multiprotein signature (n = 18) indicated that CD38(-/-) and WT mice could be segregated in response to CFA treatment. Further analyses showed that the discriminative power to distinguish these groups was reached at protein species level and not at the protein level. Hence, the need to identify and quantify proteins at protein species level to better correlate proteome changes with disease processes. It is crucial for plasma proteomics at the low-abundance protein species level to apply the ProteoMiner enrichment. All MS data have been deposited in the ProteomeXchange with identifiers PXD001788, PXD001799 and PXD002071 (http://proteomecentral.proteomexchange.org/dataset/PXD001788, http://proteomecentral.proteomexchange.org/dataset/PXD001799 and http://proteomecentral.proteomexchange.org/dataset/PXD002071).

Use of placental growth factor and uterine artery doppler pulsatility index in pregnancies involving intrauterine fetal growth restriction or preeclampsia to predict perinatal outcomes.

AIM: The potential of uterine artery (UA) Doppler pulsatility index (PI) and maternal serum placental growth factor (PlGF) level to predict perinatal outcome was explored in pregnancies complicated by intrauterine fetal growth restriction (IUGR) or preeclampsia (PE). METHODS: This longitudinal, prospective, and case-controlled study was conducted over a period of 24 months. At-risk pregnancies involving small-for-gestational-age (SGA) fetuses, IUGR, gestational hypertension (GH), or PE were investigated, analyzing UA Doppler PI findings and maternal PlGF levels determined at the time of diagnosis (third trimester). RESULTS: UA Doppler PI and maternal serum PlGF values differed significantly in pregnancies complicated by IUGR and/or PE (vs. SGA or GH, p < 0.01). In the context of IUGR or PE, both parameters also differed significantly by perinatal outcome (adverse vs. normal, p < 0.01), although no predictive advantage over UA Doppler PI alone was conferred by adding a PlGF assay. CONCLUSION: UA Doppler PI and maternal serum PlGF determinations in the third trimester help identify pregnancies at the highest risk of adverse perinatal outcomes due to IUGR and/or PE. Although joint testing confers no predictive benefit over UA Doppler PI alone, the two diagnostics are interchangeable for this purpose.

Role of 3-dimensional power Doppler sonography in differentiating pregnant women with threatened preterm labor from those with an asymptomatic short cervix.

OBJECTIVES: To compare cervical volumes and vascularization indices using 3-dimensional power Doppler sonography among singleton pregnancies with threatened preterm labor and an asymptomatic short cervix. METHODS: Three hundred asymptomatic healthy pregnant women between 24 and 34.6 gestational weeks were prospectively scanned for cervical length. If cervical length was short, defined as below the 10th percentile for gestational age, the cervical volume and vascularization indices (vascularization index [VI], vascularization-flow index [VFI], and flow index [FI]) were measured. Women receiving any treatment for preterm delivery prevention were excluded. During this period, the same sonographic parameters were measured among patients with threatened preterm labor admitted to our institution during the first 24 hours after admission. Multiple pregnancies and fetal or maternal pathologic conditions, were exclusion criteria. Data on body mass index, maternal age, smoking, parity, family history of preterm birth, mother who was born preterm, and previous preterm birth among the pregnant women were recorded. Sonographic and medical history parameters were compared between the two groups. RESULTS: Twenty-nine asymptomatic healthy women (9.6%) had a short cervix. Additionally, 71 pregnancies with threatened preterm labor were scanned. There were no statistically significant differences between the groups in medical history parameters or cervical length. The cervical volume was smaller in threatened preterm cases (12.90 versus 17.168 cm(3); P = .005). The VI and VFI were lower in women with an asymptomatic short cervix (VI, 4.369% versus 15.939%; P < .001; VFI, 1.514 versus 4.878; P < .001). The FI was higher in the short cervix group (33.581 versus 30.311; P = .006). CONCLUSIONS: Three-dimensional transvaginal sonography reveals differences in cervical volume and vascularization indices between pregnancies with an asymptomatic short cervix and cases with threatened preterm labor, although cervical length is similar in both groups.

Ultrasonication: An Efficient Alternative for the Physical Modification of Starches, Flours and Grains.

Ultrasonic (USC) treatments have been applied to starches, flours and grains to modify their physicochemical properties and improve their industrial applicability. The extent of the modification caused by USC treatment depends on the treatment conditions and the natural characteristics of the treated matter. Cavitation leads to structural damage and fragmentation and partial depolymerization of starch components. The amorphous regions are more susceptible to being disrupted by ultrasonication, while the crystalline regions require extended USC exposure to be affected. The increased surface area in USC-treated samples has a higher interaction with water, resulting in modification of the swelling power, solubility, apparent viscosity, pasting properties and gel rheological and textural properties. Starch digestibility has been reported to be modified by ultrasonication to different extents depending on the power applied. The most important treatment variables leading to more pronounced modifications in USC treatments are the botanical origin of the treated matter, USC power, time, concentration and temperature. The interaction between these factors also has a significant impact on the damage caused by the treatment. The molecular rearrangement and destruction of starch structures occur simultaneously during the USC treatment and the final properties of the modified matrix will depend on the array of treatment parameters. This review summarizes the known effects of ultrasonic treatments in modifying starches, flours and grains.

Physicochemical Characterisation of Seeds, Oil and Defatted Cake of Three Hempseed Varieties Cultivated in Spain.

The increasing use of hempseed in food products highlights the need for a comprehensive database for scientific research and industrial applications. In food development, information about the techno-functional properties of raw materials plays a crucial role in determining the suitability of each product for specific applications. Thus, this study aims to characterise three hempseed varieties (Ferimon, Henola and Uso-31), comparing their physicochemical and nutritional compositions. Moreover, the study investigates the impact of hempseed varieties on the techno-functional, physical and thermal properties of the partially defatted hempseed flours (PDHFs) obtained from single screw pressing (SSP) oil extraction. The fatty acid and tocopherol profiles of the dehulled seeds and oil were also analysed. Significant variations in yield and physical properties were observed among hempseed varieties, influenced by genetics, adaptation to agro-climatic conditions and cultivation systems. Despite its lower yield (kg/ha), Uso-31 exhibited superior 1000-seed weight, dehulling yield and larger mean seed size (1.79 ± 0.02 mm). Hempseed oil was rich in unsaturated fatty acids, particularly linoleic (51.2-53.4 g/100 g oil) and α-linolenic (14.88-18.97 g/100 oil) acids, showing variations in γ- and α-tocopherols depending on the variety. The variety also influenced the least gelation concentration (LGC) and techno-functional properties such as water absorption capacity (WAC), emulsifying activity (EA) and emulsion stability (ES). SDS-PAGE and DSC measurements indicated the presence of 11S and 7S globulin proteins with denaturation temperatures above 87.8 °C. These findings confirm that the studied hempseed flours are valuable techno-functional and nutritional ingredients suitable for sustainable food formulations.

Increased CD38 expression in T cells and circulating anti-CD38 IgG autoantibodies differentially correlate with distinct cytokine profiles and disease activity in systemic lupus erythematosus patients.

CD38 is a multifunctional protein possessing ADP-ribosyl cyclase activity responsible for both the synthesis and the degradation of several Ca(2+)-mobilizing second messengers. In mammals, CD38 also functions as a receptor. In this study CD38 expression in CD4(+), CD8(+), or CD25(+) T cells was significantly higher in systemic lupus erythematosus (SLE) patients than in Normal controls. Increased CD38 expression in SLE T cells correlated with plasma levels of Th2 (IL-4, IL-10, IL-13) and Th1 (IL-1ß, IL-12, IFN-γ, TNF-α) cytokines, and was more prevalent in clinically active SLE patients than in Normal controls. In contrast, elevated anti-CD38 IgG autoantibodies were more frequent in clinically quiescent SLE patients (SLEDAI=0) than in Normal controls, and correlated with moderate increased plasma levels of IL-10 and IFN-γ. However, clinically active SLE patients were mainly discriminated from quiescent SLE patients by increased levels of IL-10 and anti-dsDNA antibodies, with odds ratios (ORs) of 3.7 and 4.8, respectively. Increased frequency of anti-CD38 autoantibodies showed an inverse relationship with clinical activity (OR=0.43), and in particular with the frequency of anti-dsDNA autoantibodies (OR=0.21). Increased cell death occurred in CD38(+) Jurkat T cells treated with anti-CD38(+) SLE plasmas, and not in these cells treated with anti-CD38(-) SLE plasmas, or Normal plasmas. This effect did not occur in CD38-negative Jurkat T cells, suggesting that it could be attributed to anti-CD38 autoantibodies. These results support the hypothesis that anti-CD38 IgG autoantibodies or their associated plasma factors may dampen immune activation by affecting the viability of CD38(+) effector T cells and may provide protection from certain clinical SLE features.

Impact of Starch Concentration on the Pasting and Rheological Properties of Gluten-Free Gels. Effects of Amylose Content and Thermal and Hydration Properties.

The pasting and rheological properties of starch gels from different botanical origins have been widely used to evaluate the application of these starches in pharmaceutical and food products. However, the ways in which these properties are modified by starch concentration and their dependence on amylose content and thermal and hydration properties have not been adequately established so far. An exhaustive study of the pasting and rheological properties of starch gels (maize and rice (normal and waxy in both cases), wheat, potato, and tapioca) at concentrations of 6.4, 7.8, 9.2, 10.6, and 11.9 g/100 g was performed. The results were evaluated in terms of a potential equation fit between each parameter and each gel concentration. The parameters determined for the gels at the studied concentrations were correlated with the hydration properties and thermal properties by applying principal component analysis (PCA). Wheat starch, followed by normal maize and normal rice starches, presented a greater capacity to modulate their gels' pasting and viscoelastic properties via their concentration in water. On the contrary, the characteristics of waxy rice and maize, potato, and tapioca starches were barely modified by concentration in pasting assays, but the gels of potato and tapioca showed noticeable changes in their viscoelastic properties as functions of concentration. In the PCA plot, the non-waxy cereal samples (wheat, normal maize, and normal rice) were located close to each other. Wheat starch gels were the most dispersed on the graph, which is consistent with the high dependence on the concentration of the gel shown in most of the studied parameters. The waxy starches had close positions not too distant from those of the tapioca and potato samples and with little influence from amylose concentration. The potato and tapioca samples were close to the vectors of the crossover point in rheology and peak viscosity in their pasting properties. The knowledge gained from this work allows a better understanding of the effects of starch concentration on food formulations.

Modification of the microstructure of tef [Eragrostis tef (Zucc.) Trotter] flour ultrasonicated at different temperatures. Impact on its techno-functional and rheological properties.

Tef flour comes from a nutritionally-rich ancient grain gaining increasing interest in gluten-free market. Gluten-free sources are modified by different means to improve their functionality. Ultrasound treatment (US) alters flours' structure and leads to physically modified flours with a wider application range. The aim of the present work was to evaluate the impact of US treatments of moderate treatment time, 10 min, and high concentration of the aqueous flour dispersion, 25%, on the microstructural, starch damage, apparent amylose content, techno-functional, pasting and rheological properties of two tef flour varieties, white and brown. Temperature was varied (20, 40, 45, 50, and 55 °C) to modulate the impact of sonication. US treatments led to general particle fragmentation which markedly increased starch damage and lightness (L*) values. Apparent amylose content was higher after ultrasonication, as consequence of molecular fragmentation due to cavitation. Increased starch granules' exposed area led to enhanced interaction with water, promoting the water absorption index (WAI) and swelling power (SP) of treated flours. Pasting properties showed increased pasting temperatures as well as decreased viscometric profiles with lower breakdown viscosities, indicative of starch rearrangement improved by increasing temperature. Rheological properties indicated higher consistency in gels after US treatments, with improved ability to withstand stress and lower values of tan(δ)1 reflecting a higher solid-like behavior and higher strength of the gel. Temperature was found to be a crucial variable during US treatments, showing an improved degree of modification at higher temperatures in ultrasonicated tef flours, following the same trend in both varieties.

Impact of Solubilized Substances on the Techno-Functional, Pasting and Rheological Properties of Ultrasound-Modified Rice, Tef, Corn and Quinoa Flours.

The modification of flours by ultrasound (US) treatments requires excess water to suspend the sample to be treated, which must be removed after treatment to recover the ultrasonicated flour. The aim of this study was to determine the influence that the water removal method has on the final characteristics of US-treated gluten-free flours (rice, brown tef, corn and quinoa). US treatment parameters were constant, and two water removal methods were studied: freeze-drying and centrifugation + drying. The elimination of water by centrifugation resulted in the loss of solubilized compounds from the treated flours, which led to important differences between the final characteristics of US-treated flours. Ultrasonication resulted in the reduction of flours' particle size and modification of their color parameters. Techno-functional properties were modified by US treatment, where the water removal method was more influential in whole grain samples (brown tef and quinoa). Few differences were found in thermal properties among pairs of US-treated samples, indicative that the effect caused to starch was mainly attributed to ultrasonication conditions than to the drying method. The water removal method markedly influenced the pasting properties of US-treated flours, resulting in lower profiles when freeze-drying was applied and higher profiles when flours were retrieved by centrifugation. Gels made with tef, corn and quinoa presented reduced tan(δ)1 values after sonication, while gels made with rice did not show any modification. The water removal method is a decisive step in US treatments, defining the final characteristics of the treated matter, and having a great influence in the modification attributed to ultrasonication.

Endothelial progenitor cells as biomarkers of diabetes-related cardiovascular complications.

Diabetes mellitus (DM) constitutes a chronic metabolic disease characterized by elevated levels of blood glucose which can also lead to the so-called diabetic vascular complications (DVCs), responsible for most of the morbidity, hospitalizations and death registered in these patients. Currently, different approaches to prevent or reduce DM and its DVCs have focused on reducing blood sugar levels, cholesterol management or even changes in lifestyle habits. However, even the strictest glycaemic control strategies are not always sufficient to prevent the development of DVCs, which reflects the need to identify reliable biomarkers capable of predicting further vascular complications in diabetic patients. Endothelial progenitor cells (EPCs), widely known for their potential applications in cell therapy due to their regenerative properties, may be used as differential markers in DVCs, considering that the number and functionality of these cells are affected under the pathological environments related to DM. Besides, drugs commonly used with DM patients may influence the level or behaviour of EPCs as a pleiotropic effect that could finally be decisive in the prognosis of the disease. In the current review, we have analysed the relationship between diabetes and DVCs, focusing on the potential use of EPCs as biomarkers of diabetes progression towards the development of major vascular complications. Moreover, the effects of different drugs on the number and function of EPCs have been also addressed.

PCR-Based Strategy for Introducing CRISPR/Cas9 Machinery into Hematopoietic Cell Lines.

Acute myeloid leukemia is a complex heterogeneous disease characterized by the clonal expansion of undifferentiated myeloid precursors. Due to the difficulty in the transfection of blood cells, several hematological models have recently been developed with CRISPR/Cas9, using viral vectors. In this study, we developed an alternative strategy in order to generate CRISPR constructs by fusion PCR, which any lab equipped with basic equipment can implement. Our PCR-generated constructs were easily introduced into hard-to-transfect leukemic cells, and their function was dually validated with the addition of MYBL2 and IDH2 genes into HEK293 cells. We then successfully modified the MYBL2 gene and introduced the R172 mutation into the IDH2 gene within NB4 and HL60 cells that constitutively expressed the Cas9 nuclease. The efficiency of mutation introduction with our methodology was similar to that of ribonucleoprotein strategies, and no off-target events were detected. Overall, our strategy represents a valid and intuitive alternative for introducing desired mutations into hard-to-transfect leukemic cells without viral transduction.

GrabCut-based human segmentation in video sequences.

In this paper, we present a fully-automatic Spatio-Temporal GrabCut human segmentation methodology that combines tracking and segmentation. GrabCut initialization is performed by a HOG-based subject detection, face detection, and skin color model. Spatial information is included by Mean Shift clustering whereas temporal coherence is considered by the historical of Gaussian Mixture Models. Moreover, full face and pose recovery is obtained by combining human segmentation with Active Appearance Models and Conditional Random Fields. Results over public datasets and in a new Human Limb dataset show a robust segmentation and recovery of both face and pose using the presented methodology.

Techno-Functional and Gelling Properties of Acha (Fonio) (Digitaria exilis stapf) Flour: A Study of Its Potential as a New Gluten-Free Starch Source in Industrial Applications.

Fonio (Digitaria exilis Stapf) is an ancient African cereal that represents a rich source of carbohydrate, fat, fiber, vitamins, minerals, and sulfur-containing amino acids. Processing and utilization of fonio require adequate knowledge of its structural, chemical, and nutritional characteristics. The present work evaluates the structural, techno-functional, and gelling properties of fonio and compares them to other major gluten-free cereals (rice, maize, sorghum, and millet). Fonio flour presented significantly higher water absorption index and swelling power, while it scored a lower water solubility index than the reference flours. The pasting viscosity profile of fonio was similar to that of rice, with equivalent peak viscosity but a breakdown viscosity 24% lower than rice, indicative of higher stability and resistance to shearing and heating. Rheological properties demonstrated that fonio generates gels with remarkably strong structures. At 15% concentration, fonio gel withstood stress 579% higher than those observed in the reference flours without breaking its structure. Fonio flour presented the highest gelatinization enthalpy (11.45 J/g) and a narrow gelatinization temperature range (9.96 °C), indicative of a better-packed starch structure than the other analyzed flours. The texture of the gels made with fonio showed higher firmness over the evaluated period. These combined results suggest that fonio is a suitable ingredient for gel-like food formulations.

Impact of microwave radiation on in vitro starch digestibility, structural and thermal properties of rice flour. From dry to wet treatments.

Microwave radiation (MW) is an environment-friendly technology used to physically modify flours. Rice flour was MW-treated at different moisture content (MC) (3 %, 8 %, 13 %, 15 %, 20 % and 30 %). In vitro starch digestibility was determined and related to the changes caused by MW treatment to flours' structure and thermal properties, which were influenced by MC. A reduction of 49 % and 65 % in the gelatinization enthalpy of samples treated at 20 % and 30 % MC denoted a partial gelatinization. A loss of granular crystallinity in treated samples was confirmed by XR-diffraction and FTIR, particularly at 15 %, 20 % and 30 % MC. MW promoted the formation of random-coil, α-helix and ß-turn protein structure, and the disappearance of LF-ß-sheet. Morphological differences were found between samples treated at 8 % MC (loss of polygonal structure, protein layer covering granules' surface and small holes) and 30 % MC (rounded and aggregated granules, covered with exudate amylose). In vitro starch digestibility revealed that samples treated at 20 % and 30 % MC showed 40 % and 47 % higher rapidly digestible starch, 48 % and 70 % lower slowly digestible starch and 90 % lower resistant starch than the untreated flour. Flour MC in MW-treatment allowed the modulation of structural and thermal characteristics of rice flour and consequently its starch hydrolysis rate.

Determination of puberty in gilts: contrast of diagnostic methods.

BACKGROUND: Early onset of a gilt´s puberty is needed for adequate economic performance in farms, because it indicates her reproductive performance and longevity. Therefore, an effective diagnosis is needed. Our purpose was to compare different procedures (external characteristics, blood progesterone analysis and ultrasonography diagnosis) to detect puberty in 70 gilts (Topigs TN70; 240 days old) on farm conditions. Postmortem examination was the standard reference. Multiple logistic regression analysis was used to identify which combination of independent variables (predictors) best predicts the status of gilts. RESULTS: Puberty (46/70 gilts; 65.71%) was characterized by the presence of follicles larger than 6 mm, corpus albicans, corpus rubrum, and corpus luteum (postmortem examination). Vaginal length, body condition, backfat, carcass weight and progesterone blood concentration were significantly higher in pubertal than prepubertal gilts (P < 0.05). Two types of ultrasonography equipment (DELTA and W3) were compared and performed by the same senior technician (V1). The results obtained by two technicians with different levels of experience (V1 and V2, a junior technician) using W3 were also compared. Ultrasonography provided better results than other diagnostic techniques, although the effectiveness of the ultrasonography changed with technological improvements and with increased expertise of technicians. The most accurate results were found by V1/DELTA (Nagelkerke´s R2 = 0.846; Sensitivity = 0.956; Specificity = 0.958; Positive predictive value = 0.978; Negative predictive value = 0.920; Area under ROC curve = 0.957). Results using the W3 equipment could be improved when used in conjunction with vaginal length (V1; Nagelkerke´s R2 = 0.834; Sensitivity = 0.933; Specificity = 0.958; Positive predictive value = 0.977; Negative predictive value = 0.885; Area under ROC curve = 0.972) or progesterone concentration (V2; Nagelkerke´s R2 = 0.780; Sensitivity = 0.955; Specificity = 0.826; Positive predictive value = 0.915; Negative predictive value = 0.905; Area under ROC curve = 0.970). CONCLUSIONS: Ultrasonography provided better results than other diagnostic techniques. The effectiveness of the ultrasonography changes with technological improvements and with increased expertise of technicians. Results using the W3 equipment could be improved when used along with vaginal length (V1) or progesterone concentration (V2). Accuracy parameters are a guide to choose puberty diagnosis, but the farms must also evaluate effect on gilts, ease and cost of administration.

Assessment of endothelial colony forming cells delivery routes in a murine model of critical limb threatening ischemia using an optimized cell tracking approach.

BACKGROUND: Endothelial colony forming cells (ECFCs), alone or in combination with mesenchymal stem cells, have been selected as potential therapeutic candidates for critical limb-threatening ischemia (CLTI), mainly for those patients considered as "no-option," due to their capability to enhance revascularization and perfusion recovery of ischemic tissues. Nevertheless, prior to translating cell therapy to the clinic, biodistribution assays are required by regulatory guidelines to ensure biosafety as well as to discard undesired systemic translocations. Different approaches, from imaging technologies to qPCR-based methods, are currently applied. METHODS: In the current study, we have optimized a cell-tracking assay based on DiR fluorescent cell labeling and near-infrared detection for in vivo and ex vivo assays. Briefly, an improved protocol for DiR staining was set up, by incubation of ECFCs with 6.67 µM DiR and intensive washing steps prior cell administration. The minimal signal detected for the residual DiR, remaining after these washes, was considered as a baseline signal to estimate cell amounts correlated to the DiR intensity values registered in vivo. Besides, several assays were also performed to determine any potential effect of DiR over ECFCs functionality. Furthermore, the optimized protocol was applied in combination with qPCR amplification of specific human Alu sequences to assess the final distribution of ECFCs after intramuscular or intravenous administration to a murine model of CLTI. RESULTS: The optimized DiR labeling protocol indicated that ECFCs administered intramuscularly remained mainly within the hind limb muscle while cells injected intravenously were found in the spleen, liver and lungs. CONCLUSION: Overall, the combination of DiR labeling and qPCR analysis in biodistribution assays constitutes a highly sensitive approach to systemically track cells in vivo. Thereby, human ECFCs administered intramuscularly to CLTI mice remained locally within the ischemic tissues, while intravenously injected cells were found in several organs. Our data corroborate the need to perform biodistribution assays in order to define specific parameters such as the optimal delivery route for ECFCs before their application into the clinic.